bluuebunny/crossref_metadata_2025 huggingface dataset:
Opposing roles of tyrosine kinase receptors Mer and Axl determine clinical outcome in a mouse model of nephritis
Authors: Shao Wenhai , Zhen Yuxuan
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology. Lupus nephritis remains one of the most severe manifestations of SLE, with considerable morbidity and mortality. There remains a major void in the successful management of lupus nephritis. TAM (Tyro-3, Axl and Mer) receptor tyrosine kinases play an important role in the maintenance of immune homeostasis. Mer is constitutively highly expressed on glomerular endothelial cells; Axl expression is inducible in kidney under inflammatory conditions. To investigate the functions of Axl and Mer in lupus nephritis, we induced glomerulonephritis in WT, Axl-KO, Mer-KO, and A/M-KO (Axl/Mer-double knockout) mice by injection of nephrotoxic serum (NTS). Mer-KO mice developed severe glomerulonephritis, with significantly decreased survival rate and increased blood urea nitrogen (BUN) levels as compared to WT mice given the same treatment. Two weeks after injection, necrotic cell death was evident in the glomeruli of Mer-KO mice. However, Axl-KO presented with significantly increased survival rate and reduced BUN as compared to the WT, Mer-KO and A/M-KO mice injected with the same serum. Interestingly, the A/M-KO mice developed kidney inflammation comparable to WT mice. Western blot analysis revealed significantly increased Stat3 phosphorylation and caspase-1 activation in the kidney of Mer-KO mice after NTS injection. In contrast, Axl deficient NTS-injected mice developed decreased Akt phosphorylation and Bcl-xl upregulation. The reciprocal activation of Axl and Mer receptor tyrosine kinases may have a major impact on renal inflammation. Results from these studies may lead to novel therapeutic drug development targeted to lupus nephritis.
Endothelial Protein C Receptor regulates skin gamma delta T cell functions
Authors: Ramirez Kevin P , Witherden Deborah , Ruf Wolfram , Havran Wendy L
Published: January 2023

The molecular interactions controlling gamma delta (γδ) T cell functions remains poorly understood as these cells do not express many of the costimulatory receptors known to regulate alpha beta T cell activation. Dendritic epidermal T cells (DETC) are prototypic γδ cells that promote tissue homeostasis by producing keratinocyte survival factors under steady state conditions and locally releasing epithelial cell mitogens upon stimulation by skin wounding. Endothelial Protein C Receptor (EPCR) is an MHC class I-like molecule recently demonstrated to be a TCR ligand for a subset of human γδ T cells. In the skin, EPCR is expressed by keratinocytes and has been implicated in skin wound repair in vitro. We therefore hypothesized that DETC recognize and are functionally dependent on cutaneous EPCR expression. We show that mutant mice with reduced expression of EPCR display delayed repair of experimental skin wounds. This wounding phenotype is associated with reduced cellular markers of activation and defective production of inflammatory cytokines by DETC. Conversely, EPCR is highly upregulated in normal keratinocytes proximal to wound sites. EPCR may engage DETC directly as a subset of these cells express the known EPCR binding partner, CD11b, and wound proximal DETC appear to bind a soluble form of EPCR. Collectively, our data indicates that EPCR serves a costimulatory molecule for skin-resident γδ T cells.
Retinoic acid and IFNγ induce parallel Arg1 and Nos2 gene expression in primary mouse Kupffer cells.
Authors: Mohar Isaac , Crispe Ian Nicholas
Published: January 2023

Acute inflammation is associated with classically activated macrophages, while chronic inflammation is associated with fibrosis and alternatively activated (M2) macrophages. Liver fibrosis is a common complication of chronic liver inflammation, yet the molecular signals that promote M2 Kupffer cells, resident liver macrophages, are poorly defined. In the liver, activated hepatic stellate cells (HSC) are associated with fibrosis and release retinoic acid (RA). Here, we test the hypothesis that RA polarizes Kupffer cells to an M2 state. We contrast this to activation by IFNγ or lipopolysaccharide (LPS). Kupffer cells were isolated from C57BL/6J mice (N = 2–5) by sort-purification; plated at 30,000 per well for 24 hours; and then stimulated with 100 ng/mL LPS, 100 ng/mL IFNγ, 100 μM RA, or IFNγ + RA for 24 hours. Isolated RNA was analyzed by microfluidic qRT-PCR using assays for genes characteristic of macrophage activation. LPS induced Socs3, Ptgs2, Nos2, Tnf, Icam1, Il6, Il1a, Il10, and Marco. IFNγ induced Socs3, Ptgs2, Nos2, Tnf, Icam1, Ciita, Ccl12, and Klf4; and suppressed Mrc1. RA only induced expression of Arginase-1 (Arg1). Unexpectedly, co-treatment with RA and IFNγ induced additive expression. In summary, treatment of primary Kupffer cells with RA induces Arg1; co-treatment with IFNγ and RA simultaneously induces Arg1 and Nos2; and LPS, but not IFNγ, induces Il6 gene expression. These results support the hypothesis that HSC activation may induce aspects of alternative activation in Kupffer cells and demonstrate that classical and alternative activation fates of macrophages are not always mutually exclusive.
Understanding macrophage recruitment to the stressed airspaces
Authors: Saini Yogesh , Mohammad Owais , Lewis Brandon W. , Sultana Razia
Published: January 2023

The presence of functionally competent macrophages in respiratory airspaces is critical to the normal host immune responses. To determine the effect of altered macrophage (MΦ) populations on macrophage recruitment, we employed LysM+/mTOM\mEGFP+/DTA+ mice, a model of macrophage depletion and mEGFP+ labeling. Since controlled by same promoter (Lysozyme M; LysM) and separate alleles of ROSA locus, the expression of mEGFP and DTA transgenes is simultaneously induced that facilitates mEGFP+ fluorescent labeling of DTA+ cells with LysM+ activity. Flow cytometric and confocal microscopic analyses revealed the mEGFP+ labeling of entire bronchoalveolar lavage (BAL) MΦ populations in the LysM+/mTOM\mEGFP+/DTA- mice. However, the LysM+/mTOM\mEGFP+/DTA+ had a significant increase in the number of mTOM+ MΦ populations. We hypothesized that the mTOM+ population represents freshly recruited (immature) macrophages that are functionally and phenotypically differently than the mEGFP+ MΦ. The flow cytometric analyses and confocal microscopic approach to immunophenotype mTOM+ and mEGFP+ MΦ revealed increased mTOM+ cells in the DTA+ mice. The mTOM+ MΦ exhibited monocyte-like phenotype with surface expression of CD11b and CD11c whereas entire MΦ populations (~96% are mEGFP+) in the DTA- are CD11b- and CD11c+. The gene expression analysis of mTOM+ and mEGFP+ MΦ revealed significantly altered gene expression of genes for macrophage activation, surface receptors, protease, and anti-proteases. Collectively, our approach and data collected highlights the importance of studying the mature and immature (freshly recruited) macrophage populations independently.
Local Complement production and TLR crosstalk in intestinal ischemia/reperfusion
Authors: Fleming Sherry D , Pope Michael
Published: January 2023

Intestinal ischemia/reperfusion (IR)-induced tissue damage requires crosstalk between complement components and toll-like receptors (TLR). TLR2 and TLR4 are expressed on both white blood cells and intestinal epithelial cells. In addition, complement may be produced in the local tissue as well as in the liver. We hypothesized that expression of TLRs would induce local production of complement components by circulating white blood cells and together result in IR-induced injury. To evaluate this hypothesis, we created chimeric mice by injecting wildtype bone marrow to lethally irradiated TLR2 or TLR4 deficient mice or TLR deficient bone marrow to lethally irradiated wildtype mice. The chimeric mice were subjected to intestinal IR and injury, production of complement components and TLR-mediated inflammatory products analyzed. IR-induced injury required TLR4 on the intestinal tissue whereas expression of TLR2 on either intestinal or white blood cells was sufficient for injury. In contrast, PGE2 production required TLR expression on bone marrow derived cells. TLR4 expression on non-hematopoietic tissue was sufficient for IR-induced complement C3 deposition. However, C3 was deposited on TLR2 chimeric mice if any type of wildtype cells were present, similar to intestinal injury. Together these data indicate distinct crosstalk between the specific TLRs and C3 during IR-induced injury and inflammation.
Cannabinoid receptor 1 mediates M1 type macrophage polarization by G(α)i/o/RhoA/NF-κB p65 signaling pathway in murine liver injury
Authors: Li Liying , Tian Lei , Yang Le
Published: January 2023

Macrophage, as an essential component of innate immunity, has emerged as a key role in liver injury. There are two main subtypes of macrophages in liver, referred to as tissue-resident Kupffer cells and bone marrow (BM)-derived monocyte/macrophages (BMMs). Macrophages are phenotypically heterogeneous and broadly characterized according to their polarization as M1/M2 types. Our previous research had demonstrated that cannabinoid receptor 1 (CB1) mediated infiltration and activation of BMMs in mouse liver injury. Here we investigated the role of CB1 in macrophage polarization. ICR mice were lethally irradiated and received BM transplants from enhanced GFP transgenic mice. Four weeks later, mice of BM reconstruction were subjected to carbon tetrachloride (CCl4)-induced liver injury. Expressions of M1 gene signatures (CD86, iNOS, IL-1b, IL-6, TNFa) in livers or cultured primary BMMs were measured by RT qPCR or Western blot. In the chimeric murine model, we found that amount of M1 type macrophages were increased from 1 to 28 days in CCl4 treatment with a maximal increase at 28 days by flow cytometry. Almost 70% of M1 type macrophages were from BMMs (CD86+EGFP+) and 30% of them were from Kupffer cells (CD86−EGFP+). Meanwhile, the dynamic changes of M1 gene signatures were consistent in injured livers by RT qPCR or Western blot. Blockage of CB1 by administration of its specific antagonist or siRNA in vivo decreased the amount of M1 type of BMMs origin, but showed little effect on that of Kupffer cells. Activation of CB1 by its agonist, ACEA, promoted M1 polarization of BMMs, depending on G(a)i/o/RhoA/NF-κB p65 signaling pathway. In conclusion, these data suggest that CB1 plays a crucial role in regulating M1 polarization of BMMs in liver injury.
Dectin-1 restores macrophage anti-Cryptococcus neoformans activity
Authors: TAVARES ALDO , BOCCA ANAMELIA
Published: January 2023

The interaction between macrophages and Cryptococcus neoformans (Cn) can result in fungal killing or intracellular replication. The macrophage optimal response requires the specific recognition of fungal molecular structures by a variety of pattern-recognition receptors (PRRs) receptors, mainly TLRs and CLRs.. We report that zymosan (b-glucan)-stimulated macrophages enhanced the antifungal activity against Cn. This activity depends primarily on Dectin-1 activation, with minor contribution of complement receptor 3 and/or TLR2. b-glucan-stimulation also up-regulates macrophage genes associated with an effective immune response, activation of caspase 1, enhanced IL-1b secretion and phagossome acidification. Hence, macrophage permissiveness to Cn intracellular replication is a consequence of interference by the capsule in the recognition between cell wall antigens and PPRs. Our results reveal a new role for the capsule in virulence by establishing that it prevents macrophage activation and thus prevents control of intracellular infection. Therapeutic approaches that promote macrophage activation through stimulation of Dectin-1 may be useful as adjunctive therapy for cryptococcosis.
High levels of PGE2 in bone marrow transplant mice impairs autophagy in alveolar macrophages
Authors: Martinez-Colon Giovanny Joel , Moore Bethany B
Published: January 2023

Bone marrow transplantation (BMT) is commonly used as a therapeutic strategy to treat different health disorders such as cancer. Although it benefits the host by restoring a damaged immune system with a healthy immune system, BMT recipients develop different complications in handling pulmonary pathogens. Previous studies have shown that immune and structural cells in the lungs of BMT recipients highly express cyclooxygenase (COX)-2, leading to overproduction of immunosuppressive prostaglandin E2 (PGE2) and may explain the susceptibility observed in BMT recipients to opportunistic pathogens such as Pseudomonas aeruginosa. Alveolar macrophages reside in the lower airways where they compose the first line of host defense against a wide array of pathogens. Previous studies have demonstrated that alveolar macrophages can target and clear pathogens such as Pseudomonas aeruginosa via autophagy, a mechanism by which pathogens are encapsulated in an autophagosome wherein the bacterial components are targeted for degradation. We have evidence that BMT recipient mouse alveolar macrophages harbor a deficiency in autophagy. To determine if PGE2 regulates autophagy in alveolar macrophages, we performed a series of in vitro and in vivo assays using a syngeneic BMT mouse model. Our results indicate impaired autophagy activity in alveolar macrophages in the presence of exogenous PGE2 as well as in the lung compartment of BMT mice. These observations highlight targeting the PGE2 signaling pathway as a method to restore immune activity in alveolar macrophages post-BMT.
Chronic HIV-1 infection induces the depletion and functional impairment of group-3 innate lymphoid cells through plasmacytoid dendritic cells and type I interferon
Authors: Zhang Zheng , Zhao Juanjuan , Cheng Liang , Wang Fu-Sheng , Su Lishan
Published: January 2023

Group 3 innate lymphoid cells (ILC3s) play a key role in promoting antibody production and gut inflammation and immunity; however, their characteristics and relevant regulatory mechanisms in chronic HIV-1 infection are poorly understood partially due to the lack of a robust model. We, here, reported that functional ILC3 cells are developed in all lymphoid organs in humanized mice with similar phenotypes to human counterparts. Importantly, chronic HIV-1 infection activated these tissue-resident ILC3 cells, and subsequently depleted them and preferentially impaired their IL-17a and IL-22 production in vivo of humanized mice, while anti-viral therapy efficiently reversed this alteration. HIV-1-induced depletion of ILC3s and their functional impairment could also be largely recovered by the depletion of plasmacytoid dendritic cells (pDCs) or in vivo of humanized mice and by the blockade of both interferon (IFN)-I and CD95/Fas pathway in vitro. Blockade of IFN-I also recovered IL-17a and IL-22 production in vivo of humanized mice with chronic HIV-1 infection. Additionally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. Finally, ILC3 depletion was found to be associated with loss of gut mucosa barrier in HIV-1-infected patients. Thus, we conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis.
The impact of lung infection on wound repair responses
Authors: Crane Meredith , Xu Yun , Henry William L. , Albina Jorge E. , Jamieson Amanda M.
Published: January 2023

The incidence of pulmonary infection is elevated in patients with traumatic injury, and the severity of disease, morbidity, and mortality is positively correlated with the degree of trauma. While this clinical problem is well documented, its mechanistic basis is not well understood. Current models are designed to assess the effect of trauma on the development of pneumonia, but lack the ability to measure the subsequent effect of lung infection on wound repair. We have established a model that allows for the simultaneous assessment of wound healing and lung responses in mice with surgical wounds and Influenza A virus (IAV) infection. Mice are wounded by the subcutaneous implantation of sterile polyvinyl alcohol (PVA) sponges or by excision of tail skin and infected with IAV 24 hours later. PVA sponge implantation allows for the assessment of cellular and cytokine responses to injury, while excisional tail wounding allows for the measurement of rate of repair. Our data demonstrate that IAV infection results in delayed wound repair. Furthermore, the wounds of infected mice have lower repair cytokine concentrations and decreased cellularity, mediated in part by impaired trafficking of Ly6Chi monocytes. Wounded mice with IAV infection also have elevated concentrations of proinflammatory cytokines and chemokines in the serum and bronchoalveolar lavage fluid, suggesting an inflated systemic inflammatory response. Taken together, these data indicate that the presence of viral lung infection impairs the normal progression of wound repair. Future studies will determine whether this effect is specific to viral infection, or whether other lung injuries including pulmonary bacterial infection have a similar effect on wound repair responses.
Natural killer cell dysfunction in HIV-exposed uninfected infants correlates with acquisition of lower respiratory tract infection
Authors: Smith Christiana E , Jalbert Emilie , de Almeida Volia , Mussi-Pinhata Marisa , Cohen Rachel A. , Yu Qilu , Amaral Fabiana , Pinto Jorge , Alarcon Jorge , Siberry George , Weinberg Adriana
Published: January 2023

Background

HIV-exposed uninfected infants (HEU) have higher rates of severe and fatal lower respiratory tract infections (LRTI) than HIV-unexposed infants (HUU), but their antibody responses to vaccines and infections do not differ. We hypothesized that natural killer cells (NK) are impaired in HEU and may predispose them to severe LRTI.

Methods

Peripheral blood mononuclear cells (PBMC) from Brazilian HEU and HUU were cryopreserved at birth and 6 months. NK phenotype and function assessed by flow cytometry after overnight incubation with and without K562 cells were compared among HEU who developed LRTI in the first 6 months of life (LRTI+), HEU who did not (LRTI−) and HUU, using Mann Whitney tests.

Results

The proportion of NK among PBMC was lower in 13 HEU vs. 22 HUU at birth (1.7% vs. 10.3%, p<0.0001) and in 51 HEU vs. 72 HUU at 6 months (3.1% vs. 4.6%, p=0.0008). The proportion of stimulated NK that upregulated IFNγ (after subtraction of unstimulated controls) was lower in HEU vs. HUU at birth (0.8% vs. 2.6%, p=0.03) and at 6 months (4.1% vs. 8.5%, p=0.0008). In addition, 6 LRTI+ and 7 LRTI− HEUs differed at birth by the following: 1) K562-specific lysis was lower in LRTI+ vs. LRTI− HEU (5.6% vs. 9.9%, p=0.046); 2) The proportion of stimulated NK that upregulated IFNγ was lower in LRTI+ HEU vs. HUU (0.6% vs. 2.6%, p=0.01), but not in LRTI− HEU (2.5%); 3) Perforin upregulation of stimulated NK was absent in LRTI+ HEU (−5.9%), but present in HUU (4.6%, p=0.01 vs. LRTI+ HEU) and in LRTI− HEU (2.5%). At 6 months, there were no differences between LRTI+ and LRTI− HEU.

Conclusion

NK numbers and function are impaired in HEU. Poor NK function at birth is associated with acquisition of LRTI in the first 6 months of life and may predict infectious complications in HEU.
Novel regulation of ILC by memory CD4 T cells during protective responses against influenza A virus
Authors: Strutt Tara M , Hwang Ji Hae , Swain Susan L.
Published: January 2023

The primary goal of vaccination is to protect individuals from the morbidity and mortality of pathogen exposure by generating immunological memory. Our current understanding is that memory CD4 T cells, commonly regarded as the regulators of memory cytotoxic CD8 T cells and/or antibody-producing B cells, provide protection by hastening pathogen clearance through ‘faster, bigger, and better’ immune responses. It is now clear, however, that many different subsets of CD4 T cells, many with specialized functions other than providing ‘help’, are generated during immune responses against pathogens. In studies designed to ascertain the full functional potential of memory CD4 T cells responding against influenza A virus (IAV), we found that virus induced TH1-like, as well as in vitro polarized TH1 or TH17 memory CD4 T cells, enhance early innate inflammatory responses that correlate with better and earlier control of IAV in infected lungs. In further studies, we found innate lymphoid cells (ILC), which are not normally associated with anti-viral responses but rather associated with healing and wound repair, are also mobilized by memory CD4 T cells during the early stage of the response against IAV. Unexpectedly, the protection afforded by memory CD4 T cells against lethal IAV challenge is significantly compromised when ILCs are depleted. These findings reveal a previously unappreciated beneficial role of memory CD4 T cells in regulating tissue homeostasis during recall responses against pathogens through the regulation of ILC subsets.
Genomic modifiers of natural killer cells, immune responsiveness and lymphoid tissue remodeling together increase host resistance to viral infection
Authors: Teoh Jeffrey , Gillespie Alyssa L , Lee Heather , Prince Jessica , Stadnisky Michael D , Anderson Monique , Nash William T , Rival Claudia , Wei Hairong , Gamache Awndre , Farber Charles , Tung Kenneth S K , Brown Michael G
Published: January 2023

The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust Dk-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.
A systems biology approach unravels the host response to West Nile virus infection in human dendritic cells
Authors: Bowen James R , Suthar Mehul S
Published: January 2023

West Nile virus (WNV) is a neurotropic Flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Previous studies in mice have found that dendritic cells (DCs) are pivotal for viral control and programming of antiviral immunity in a RIG-I like receptor (RLR) and type I interferon (IFN) dependent manner. To gain a better understanding of human WNV infection, we evaluated infection of human monocyte DCs (moDCs). We found that moDCs support productive WNV infection and that stimulation of RLR and type I IFN signaling potently restricts viral replication. Most intriguingly, we observed significant variability in viral infection of moDCs generated from different donors, ranging from 6–56% of cells infected by 48 hours post infection. To define the antiviral landscape during WNV infection, we performed mRNA sequencing and found that, in contrast to RLR and type I IFN signaling, WNV infection partially triggers expression of antiviral defense response related genes. Despite strong induction of IFNβ during WNV infection, we observed minimal expression of cytokines that promote antiviral T cell immunity (IL-4, IL-12, IL-7, IL-15) and costimulatory molecules (CD80, CD86, CD40) at the transcriptional and protein level. Using weighted gene co-expression network analysis combined with cis-regulatory sequence analysis, we identified STAT5 as an important regulatory node downstream of RLR signaling. Most intriguingly, we found that STAT5 was not induced during WNV infection, suggesting that WNV dampens DC activation by preventing STAT5 activation. Further studies are underway to better understand how RLR signaling promotes STAT5 activation and to define the viral factors that regulate STAT5 activation.
Ly6Chi monocytes control acute Ross River virus infection through an IRF7-dependent mechanism that can be counteracted by determinants in nsP1
Authors: Haist Kelsey Cornell , Burrack Kristina S , Diamond Michael S , Suthar Mehul S , Morrison Thomas E
Published: January 2023

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause large epidemics of debilitating musculoskeletal disease. Monocytes have been implicated in the pathogenesis of these infections; however, their specific roles are not clearly defined. To investigate the role of inflammatory Ly6Chi CCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administering diphtheria toxin (DT). Compared to DT-treated WT mice, DT-treated CCR2-DTR+ mice exhibited reduced weight gain and more severe disease signs following RRV infection. DT-treated CCR2-DTR+ mice had reduced numbers of monocytes in circulation and musculoskeletal tissues compared to control mice. Further supporting a role for inflammatory monocytes in control of acute alphavirus infection, depletion of CCR2+ cells restored virulence and increased viral loads in musculoskeletal tissue of mice infected with RRV encoding attenuating mutations in nsP1 to levels in monocyte-depleted mice infected with fully virulent RRV. A similar increase in viral loads was observed in tissues of DT-treated CCR2-DTR+;Rag1−/− mice infected with the nsP1 mutant virus. Furthermore, adoptive transfer of WT, but not Irf7−/−, Ly6Chi monocytes into DT-treated CCR2-DTR+ mice reversed the increased viral loads of the nsP1 mutant virus. In vitro, co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was MAVS- and IRF3/IRF7-dependent. Collectively, these data suggest that IRF7-dependent functions of monocytes are critical for control of acute alphavirus infection, and that determinants in nsP1 may counteract these responses.
The emerging role of apoptosis-associated speck-like protein containing a CARD (ASC) in dendritic cell metabolism and function.
Authors: McKeithen Danielle N , Omosun Yusuf , Ryans Khamia , Simoneaux Tankya , Ananaba Godwin , Eko Francis , Black Carolyn , Igietseme Joseph , Blas-Machado Uriel , He Qing
Published: January 2023

Chlamydia trachomatis (C. trachomatis) is a major infectious bacterial agent that causes sexually transmitted infections (STIs) worldwide. The regulatory functions of dendritic cells (DCs) play a major role in protective immunity against Chlamydia infections. The mechanism(s) underlying this immunomodulation are not fully understood. The inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) regulates the direction of immunity against such bacterial infections. In this study, we assessed the effect of ASC on the metabolism and function of Chlamydia infected DCs. Our results revealed that mitochondria of infected ASC−/− DCs had a gradual disruption of the cristae compared to the normal narrow pleomorphic cristae of uninfected wild type (WT) DCs and ASC−/− and infected WT DCs. In addition to the damaged mitochondria, we observed ASC−/− DCs had significantly reduced pyruvate production; the output of the metabolism of glucose known as glycolysis, following Chlamydia infection. Finally, the DCs from Chlamydia infected ASC−/− mice also displayed defective activation and antigen presentation. Our results suggest that ASC may play a role in cellular respiration of Chlamydia infected DCs, starting from glycolysis to the electron transport chain (ETC) which occurs within the mitochondria, thereby effecting the actions and functions of these DCs.
Transgelin-2 mimics bacterial SipA and promotes membrane ruffling and phagocytosis in lipopolysaccharide-activated macrophages
Authors: Jun Chang-Duk , Kim Hye-Ran
Published: January 2023

Some bacteria utilize their secreted proteins to induce massive actin polymerization at the site of invasion in host cells. Salmonella protein SipA polymerizes host actin under low ionic conditions, where it is usually completely suppressed, and induces membrane ruffling as an infection mechanism. However, no protein that possesses SipA function has been identified in eukaryotic cells. We show here that transgelin-2 – which reveals a similar function to SipA in vitro – is dramatically induced in macrophages in response to lipopolysaccharide via partially the NF-κB pathways. Transgelin-2 intensively localizes at the actin-rich membrane ruffles during fMLP stimulation. Interestingly, macrophages with trangelin-2 deficiency (TAGLN2−/−) reveal a dramatic reduction of membrane ruffles with attenuated membrane dynamics, resulting in retarded phagocytic functions. Collectively, these results suggest that host macrophages have evolved to utilize the same mechanism of bacterial infection to engulf invaded bacteria.
Resident memory CD8+ T cells in the liver require presentation by non-parenchymal cells to differentiate
Authors: Bertolino Patrick J , Wong Yik Chun , Sierro Frederic , Lu Bo , Tay Szun Szun , Wood Nicole A. W. , McGuffog Claire , Alexander Ian E. , McCaughan Geoffrey W. , Bowen David G.
Published: January 2023

Tissue-resident memory T cells (TRM) have been identified in different organs including skin and lung. Whether TRM can differentiate in the liver is unknown. To induce intrahepatic CD8+ T cell responses, recipient mice were transferred with anti-OVA transgenic T cells and treated with recombinant adeno-associated viral vectors (rAAV) that target mouse hepatocytes to induce de novo OVA expression in the liver. We have recently shown that a low rAAV dose induced a low antigen load and allowed the development of intrahepatic effector T cells. We investigated here whether these cells differentiated into TRM.

When 1–5% of hepatocytes expressed OVA, functional memory OVA-specific CD8+ T cells expressing CD69, a common TRM marker, were established in the liver. Intra-vital multi-photon microscopy experiments revealed that these memory cells moved slowly and against the blood flow, suggesting that they were patrolling the hepatic sinusoids. To test the ability of these cells to recirculate, livers containing memory T cells were transplanted into naive recipients. Most donor memory CD8+ T cells were detected within the transplanted livers and very few recirculated into lymphoid tissues, suggesting that they were liver resident. Importantly, when OVA presentation was restricted to hepatocytes, naïve CD8+ T cells became effectors but failed to develop into TRM.

In summary, low numbers of antigen-expressing hepatocytes promote the differentiation of liver CD8+ TRM. Although hepatocytes are critical in providing antigen, the differentiation of intrahepatic CD8+ TRM requires non-hepatocyte antigen-presenting cells. Our results have important implications for vaccine development and for the treatment of hepatotropic infections.
The generation of CD8+CD103+ tissue resident memory cells is decreased in the brain of B7-H1 knockouts after virus infection
Authors: Pavelko Kevin D , Dong Haidong
Published: January 2023

The generation of CD8+ tissue resident memory T-cells (TRM) ensures rapid recognition of viruses after secondary infection and is important for long term defense against tumor formation. We find that intracranial infection with Theiler’s murine encephalomyelitis virus (TMEV) generates a robust CD8+ effector response after acute infection. This response resolves with virus clearance and only CD8+CD103+ brain TRM remain. Much is known about the development of acute T-cell responses to TMEV, however less is known about how TRM are generated and maintained within the brain. Previously, we found that B7-H1 prevents the entry of effector CD8+ T-cells into the memory pool. In the current studies we explore the role of B7-H1 in the development and maintenance of brain TRM. We find that B7-H1 expression within the host tissues is important for maintaining TRM in the brain after TMEV infection. By studying acute and long-term post-infected B7-H1 knockout and wild-type mice we find that B7-H1 contributes to the formation and maintenance of TRM. The expression of B7-H1 is required within the host tissues to promote this memory population as the absence of B7-H1 on T-cells did not alter TRM formation. In addition, we find an increase in CD103 negative antigen non-specific CD8+ PD1+ T-cells in B7-H1 knockout hosts, a population previously identified as CD8+ regulatory T cells. This suggests that B7-H1 may also control this population of tissue resident CD8+ regulatory T cells (TRR). These findings will be important for further understanding the PD1/B7-H1 axis and its role in TRM and TRR development and for understanding how therapies that target these molecules can be optimized to promote T-cell memory formation and maintenance when used as therapy.
Age-associated oxidation of innate immune proteins in the lung mucosa alters the host response to Mycobacterium tuberculosis
Authors: Moliva Juan I. , Sasindran Smitha J. , Rajaram Murugesan V.S. , Schlesinger Larry S. , Turner Joanne , Torrelles Jordi B.
Published: January 2023

Age-associated immune changes are a risk factor for the development of tuberculosis (TB); however the phenomenon of inflammaging, a state of heightened systemic inflammation observed in the elderly, particularly within the lung mucosa has not been addressed. Mycobacterium tuberculosis (M.tb) infection results in bacterial deposition in the lung alveoli where they are bathed in alveolar lining fluid (ALF). ALF contains soluble innate factors that mediate the M.tb-host cell encounter and subsequent immune response. Recent studies indicate that local inflammation and oxidation occur within the ALF. We hypothesize that ALF components lose their function as we age, and this deficiency favors the establishment of M.tb infection. Previously, we reported increased oxidation of proteins within ALF from human adult (ages 25–44) and elderly (ages >65) populations, in addition to elevated basal levels of inflammatory cytokines and complement components. Here, we extended our findings to explore the impact of adult or elderly ALF during M.tb infection. Although the levels of SP-A and C3 were elevated in elderly ALF, their binding to M.tb was reduced, suggesting a defect in innate immune function with age. To evaluate the impact of ALF age-associated changes in vivo, mice were infected with M.tb pre-exposed to adult or elderly ALF. Mice infected with elderly ALF-M.tb had significantly increased bacterial growth within the lung and spleen compared to adult ALF-M.tb infected mice, suggesting that loss of innate immune function within ALF could have long term consequences in controlling M.tb infection. We conclude that M.tb may benefit from the declining host defense function of the lung mucosa in the elderly, driving host susceptibility to TB.
Bone loss in HLA-DRβ1-bearing humanized mice following oral challenge with Porphyromonas gingivalis
Authors: Brand David D , Sandal Indra , Luo Jiwen , Prislovsky Amanda , Whittington Karen , Dong Chen , Rosloniec Edward , Karydis Anastasios
Published: January 2023

Periodontal disease (PD) is a chronic pro-inflammatory response to oral pathogens such as Porphyromonas gingivalis which culminates in alveolar bone loss and loss of dental structure and function. Like rheumatoid arthritis (RA), periodontal disease is influenced strongly by lifestyle factors such as tobacco use, but also greatly influenced by genetic components, such as the “shared epitope,” which refers to a conserved linear sequence of amino acids in the DRβ1 chain of the HLA-DRα/β heterodimer between amino acids 67 and 74. Aggressive PD (the most destructive form of PD) is also now thought to be associated with specific HLA haplotypes including DRβ1. Using IL-17Frfp and Foxp3gfpreporters engineered into I-A° C57BL/6 mice expressing chimeric mouse/human HLA-DRβ1 (B6.DR1 mice), we find that gingival brushing with P gingivalis results in a rapid Th17 response in the peripheral blood and cervical lymph nodes, which decreases shortly after brushing. Evidence of a chronic infection can be detected in these nodes three months after oral inoculation. In addition to Th17 responses, we also find a concomitant increase in Th17-related cytokine production that parallels the Th17 activity. Furthermore, we measured a brisk production of antibodies directed against P. gingivalis and also against cyclic citrullinated peptides (these ACPAs are widely used as the principle early diagnostic for RA) as well as a loss in trabecular bone that can be detected in peri-articular bone structures, e.g. distal to the alveolar bone structures normally measured in PD models. Our findings suggest that periodontal disease can have far-reaching effects in bone morphology which may be driven by Th17/cytokine/ACPA activity even in the absence of other pathologies.
Determining transcriptional networks regulating the response of alveolar macrophages to Mycobacterium tuberculosis
Authors: Rothchild Alissa , Amon Lynn , Mai Dat , Aderem Alan
Published: January 2023

Mycobacterium tuberculosis (Mtb) continues to be a major global health problem, infecting nearly one-third of the world’s population and leading to around 1.5 million deaths each year. Current drug regimens last 6–9 months and there is still no effective vaccine for adults. It is still not fully understood how innate immune cells first respond to Mtb within the lung.

Alveolar macrophages (AMs) are the first cells in the lung to become infected with Mtb after aerosol exposure. Therefore, these macrophages play a crucial role in establishing the initial inflammatory response to Mtb and the priming environment of the adaptive response. Previously, technical limitations in cell isolation and RNA sequencing restricted gene expression studies of Mtb-infected macrophages to in vitro culture systems. These studies may have missed crucial signaling pathways unique to the immunoregulatory lung environment surrounding AMs.

To investigate the in situ response of AMs to Mtb, we developed a high-dose aerosol murine infection model with fluorescent-tagged bacteria to isolate Mtb-infected AMs at early time points after infection by fluorescence-activated cell sorting. We have now measured the global gene expression profiles of both naïve and Mtb-infected AMs by RNA-seq. Preliminary data has revealed numerous regulatory and immune pathways that are altered in response to Mtb infection. Pathway enrichment analysis identified the oxidative stress response as one of the most altered pathways and promoter scanning has identified Nrf2, a master transcription factor of the oxidative stress response, as a predicted regulator. Further network analysis of these pathways and their effects on subsequent disease outcome is currently being undertaken.
Interferon-gamma and Effector Cell Activation are Required for Antibody-Mediated Immunity against Genital Chlamydia Infection
Authors: Naglak Elizabeth Kathryn , Morrison Sandra G. , Morrison Richard P.
Published: January 2023

Defining the mechanisms of immunity conferred by the combination of antibody and CD4+ T cells is fundamental to designing an efficacious chlamydial vaccine. Using the Chlamydia muridarum genital infection model of mice, which replicates many features of human C. trachomatis infection and avoids the characteristic low virulence of C. trachomatis in the mouse, we previously demonstrated a central role for antibody in immunity to chlamydial infection. We found that antibody alone was not protective. Instead, protection appeared to be conferred through an undefined antibody-cellular interaction. Using gene knockout mice and in vivo cellular depletion methods, antibody-mediated protection was shown to be solely dependent on the activation of an effector cell population in genital tract tissues by CD4+ T cells. Furthermore, the CD4+ T cell-secreted cytokine interferon-gamma (IFNγ) was found to be a key component of the protective antibody response. The function of IFNγ was not related to the immunoglobulin class or magnitude of the Chlamydia-specific antibody response or to recruitment of an effector cell population to genital tract tissue. Rather, IFNγ appears to be necessary for activation of the effector cell population that functions in antibody-mediated chlamydial immunity. Ongoing studies aim to identify the effector cell population(s) involved in the antibody-mediated protective response. Our results confirm the central role of antibody and CD4+ T cells in immunity to chlamydia reinfection, and demonstrate a key function for IFNγ in antibody-mediated protection.
Expression of human hTLR4/MD2 in vivo in mice alters infection with C. burnetii
Authors: Robison Amanda , Hedges Jodi F , Kimmel Emily , Hajjar Adeline M. , Jutila Mark A.
Published: January 2023

Human and mouse TLR4/MD2 molecules respond differently to specific types of LPS. Their response to normally acylated LPS is similar, but they respond differently to hypoacylated LPS found on specific gram-negative bacteria. The LPS of Coxiella burnetii is hypoacylated, heavily glycosylated and is known to cause a minimal response by human cells. We hypothesized that mice expressing human TLR4/MD2 (hTLR4/MD2) molecules would respond differently to C. burnetii infection compared to wild type mice. Mice expressing hTLR4/MD2 molecules had enhanced weight loss, and more bacteria in tissues than did wild-type mice. Bone marrow chimera experiments indicated that mice expressing hTLR4/MD2 on stromal/epithelia cells had more severe disease and enhanced bacterial replication compared to when mouse TLR4 was expressed on these cells. Surprisingly, hTLR4 expression on hematopoietic cells was of minimal consequence. These data suggest that species-specific TLR4/MD2 responses in stromal and epithelial compartments can alter the disease course. Studying C. burnetii infection in hTLR4/MD2-expressing mice will result in findings that are likely more readily applicable to human patients.
The N terminus of Yersinia pestis’ YscF masks YscF’s naturally occurring PAMP, inhibiting inflammation and allowing for colonization in vivo
Authors: O’Donnell Kyle Lee , Alvine Travis D , Osei-Owusu Patrick , Niles Matthew , Bradley David
Published: January 2023

Type III secretion (T3S) systems, common to many Gram- bacteria, act as a needle to deliver virulence factors directly into eukaryotic cells, often modulating host immunity. Multimers of YscF polymerize to form the surface-located T3S needle-like structure. We have previously shown that YscF has PAMP properties, acting as both a TLR2 and TLR4 agonist. We have also shown that modulation of this PAMP activity in vitro is located in the N terminus of YscF. We hypothesized that the N terminus of YscF contributes to Y. pestis survival within the eukaryotic host. We generated a 15 amino acid N terminal truncation of YscF (delta15YscF) expressed in Y. pestis. The truncation did not affect T3S system translocation. The delta15YscF Y. pestis strain was able to secrete Yops, albeit at a level lower than wild type (wt) Y. pestis. As expected, wt Y. pestis colonized the spleen and liver, whereas delta15YscF Y. pestis exhibited significantly impaired colonization in both organs. Mice infected with delta15YscF Y. pestis developed significantly more inflammation compared to wt Y. pestis. At 3 days p.i., delta15YscF spleen weights were significantly heavier and spleen inflammation was accompanied by a significant enhancement of macrophage and neutrophil migration compared to wt mice. These data support a role for the N terminus of YscF contributing to host bacterial clearance. Truncating the N terminus may result in a PAMP which is thereby unmasked and able to induce an inflammatory response.
Multi-step Th17 differentiation in response to segmented filamentous bacteria in the mouse intestine
Authors: Sano Teruyuki , Yang Yi , Diehl Gretchen , Chen Alessandra , Kaplan Daniel H , Littman Dan R
Published: January 2023

Th17 cells have significant roles in maintaining homeostasis and regulating host defense against various pathogens in our bodies. Our laboratory initially identified segmented filamentous bacteria (SFB) as a unique commensal that is sufficient for Th17 cell differentiation and promotion of Th17-dependent autoimmune diseases such as a mouse model of spontaneous arthritis. The molecular and cellular requirements of SFB-induced Th17 cell differentiation are still unclear. To understand the whole process of Th17 cells differentiation in vivo, we developed SFB-specific T cell receptor transgenic (7B8) mice. We can trace SFB-specific Th17 cell differentiation and response by transferring fluorescently-labeled 7B8 naïve T cells into SFB-gavaged host mice. Using this approach, we have elucidated the requirements for cytokines and antigen presenting cells (APCs) to understand the process of Th17 cell differentiation. Here, we describe that initial induction and expansion of Th17 cells occurs in mesenteric lymph nodes (MLN), and their subsequent migration to intestine is integrin β7-dependent. Although RORγt expression in Th17 cells is mainly dependent on IL-6 signaling in the MLN, IL-23R signaling also contributes to RORγt expression in Th17 cells in the ileum in the absence of IL-6. CD103+ CD11b+ APCs are not important for induction and initial expansion of SFB-specific Th17 cells in MLN, but play important roles in maintenance of SFB-specific Th17 cells in the ileum. Taken together, these results indicate that Th17 cell differentiation proceeds in multiple discrete stages.
Commensal metabolite indol-3-propionic acid promotes gut barrier function by regulating IL-22 production during intestinal inflammatory conditions
Authors: Romagnoli Pablo A , Shenk Gregory K , Pham Quynh-Mai , Maher Leigh , Khanna Kamal Mohan
Published: January 2023

Microbiota-derived metabolites provide signals to maintain homeostasis of the epithelial cell layer in the intestinal mucosa that in turn shape immune responses to bacterial pathogens. In contrast, disruption of these signals can result in dysregulated responses to microbiota that cause inflammatory bowel diseases (IBD). Here we tested the hypothesis that a metabolite produced via tryptophan metabolism by commensal bacteria, Indol-3-propionic acid (IPA), promotes immune homeostasis and gut barrier function in the intestinal mucosa during inflammatory conditions and prevents IBD. We investigated the effect of IPA on the intestinal mucosa using a model of colitis caused by oral infection with Citrobacter rodentium (Crod). Remarkably, we observed that IPA treatment results in protection of mice from colitis. Our studies show that IPA treatment results in increased expression of IL-22 by intraepithelial lymphocytes (IELs) of the small intestine (S). In fact, most of the IL-22 observed at 4 days following oral Crod infection is produced by IELs from small intestines. Since we have previously shown that IPA is a potential ligand of the nuclear receptor pregnane X receptor (PXR), which contributes to the maintenance of the intestinal barrier function, we further investigated if IPA protects by activating PXR or other pathways in the gut mucosa. Indeed we discovered an increase in the absolute numbers of IL-17A and IFNγ producing lymphocytes from the IEL compartment of the colon by the IPA treatment and this effect was dependent on PXR expression. In conclusion, we believe the discovery of metabolites like IPA that promotes the repair of the intestinal epithelial cell layer can provide new strategies to prevent and treat IBD.