bluuebunny/crossref_metadata_2025 huggingface dataset:
RSV inhibits myeloid cell recruitment during S. aureus coinfection
Authors: Rich Helen Eva Adams , Morris Susan H , Lukacs Nicholas W , Moore Bethany B
Published: January 2023

Respiratory syncytial virus (RSV) infects almost all humans by the age of 2. RSV more often causes severe lower respiratory tract disease in younger children and older adults, which may be exacerbated by increased susceptibility to secondary bacterial infection (coinfection). Like influenza, it is thought that RSV may predispose the lung to opportunistic infections with strains of bacteria normally present as colonizers of the nasopharynx, predominantly Staphylococcus aureus and Streptococcus pneumoniae. While there has been significant investigation into mechanisms of RSV/bacterial coinfection, models that faithfully replicate the pathology of human RSV infection have not been fully explored. To examine this aspect, we infected mice with a strain of RSV (rA2-line 19F, 2×105 PFU) that induces significant IL-13, mucus, and airway hyperresponsiveness. We found that RSV infection six days prior to S. aureus infection (5×107 CFU) reduces bacterial clearance. Flow cytometry data suggest that RSV infection alters cellular recruitment in response to S. aureus, largely by reducing neutrophils and monocytes. Surprisingly, we saw increased levels of the monocyte chemokine CCL2, suggesting compensatory chemokine production in response to dampened cellular recruitment. Additionally, we observed no change in several cytokines that exacerbate inflammation during influenza/S. aureus coinfection, including IL-1β, IL-6, and TNFα. Together, these data suggest a markedly different mechanism of increased susceptibility to bacterial infection during RSV, characterized by reduced--rather than overexuberant--myeloid cell recruitment.

Supported by grants from NIH (R35HL144481 to Beth Moore, T32HL007517 to Helen Rich) and the Michigan Postdoctoral Pioneer Program at the University of Michigan Medical School.
Baseline NK cell frequencies are greater in individuals with asymptomatic SARS-CoV-2 infection compared to those that develop symptomatic illness
Authors: Graydon Elizabeth , Lindrose Alyssa , Anderson Stephen , Malloy Allison , Lizewski Stephen , Lizewski Rhonda , Weir Dawn , Goforth Carl , Sun Peifang , Letizia Andrew , Mitre Edward
Published: January 2023

As NK cells are critical for control of viral infections, we tested the hypothesis that individuals with asymptomatic SARS-CoV-2 infection have greater NK cell frequencies prior to infection than individuals with mild, symptomatic infection. Marine recruits who elected to participate in the COVID-19 Health Action Response for Marines (CHARM) study were prospectively followed for two months twice weekly using PCR testing for SARS-CoV-2 infection and symptom questionnaires. Peripheral blood mononuclear cells obtained prior to infection during enrollment in the study and at various time points post-infection from 47 participants diagnosed with asymptomatic infection and 49 with symptomatic infection were analyzed by flow cytometry for frequencies of total NK cells, NK cell subsets, and surface expression of activating and inhibitory receptors. Consistent with our hypothesis, frequencies of total (CD3− CD14− CD19− CD56+) NK cells were significantly greater at baseline in asymptomatic compared to symptomatic participants (10.4% vs 8.3%, p = 0.015). This difference was due to increased frequencies of mature (CD56dim CD16+) NK cells in asymptomatic individuals (76.3% vs 69.8%, p = 0.049). No differences were observed in baseline frequencies of immature (CD56bright CD16−) or adaptive (CD56dim CD16+ NKG2C+ CD57+) NK cells, or in surface expression of activating (NKG2C, NKG2D) or inhibitory (NKG2A, KIR2DL1, KIR2Dl2/L3/S2, KIR3DL1) receptors. Frequencies of total and mature NK cells decreased significantly post-infection, reaching a nadir at 3–4 weeks. In contrast, frequencies of immature NK cells increased by 4 weeks. These results suggest that NK cells may play a protective role against symptomatic SARS-CoV-2 infection.

This work was supported by a grant from the Defense Health Agency through the Naval Medical Research Center (work unit number 9700130).
Human β-defensin 2 (hBD-2) improves neutrophilia and bacterial clearance during cigarette smoke exposure
Authors: Milad Nadia , Pineault Marie , Beaulieu Marie-Josée , Aubin Sophie , Jensen Benjamin A H , Morissette Mathieu
Published: January 2023

Rationale

Treatment of cigarette smoke-associated lung diseases has largely focused on broad-spectrum anti-inflammatory therapies, such as inhaled corticosteroids. However, these therapies have recently been shown to enhance patient susceptibility to pneumonia.

Objective

Assess whether the anti-microbial peptide, human β-defensin 2 (hBD-2), can simultaneously reduce pulmonary inflammation in cigarette smoke-exposed mice while maintaining immune competence during bacterial exacerbation.

Methods

Mice were exposed to cigarette smoke acutely (4 days) or chronically (5 days/week for 7 weeks) and administered hBD-2 intranasally or by gavage. In a separate model of acute exacerbation, chronically exposed mice treated with hBD-2 were infected with non-typeable Haemophilus influenzae prior to sacrifice.

Results

In the acute exposure model, cigarette smoke-associated pulmonary neutrophilia was significantly blunted by both local and systemic hBD-2 administration. Similarly, chronically exposed mice administered hBD-2 therapeutically exhibited reduced pulmonary neutrophil infiltration and downregulated pro-inflammatory signaling in the lungs compared to vehicle-treated mice, as assessed via microarray. Finally, in a model of bacterial exacerbation, hBD-2 administration effectively limited neutrophil infiltration in the lungs while markedly reducing pulmonary bacterial load.

Conclusions

This study shows that hBD-2 treatment can significantly attenuate lung neutrophilia induced by cigarette smoke exposure while preserving immune competence and promoting an appropriate host-defense response to bacterial stimuli.

Supported by CIHR
Increased M1 macrophage activity makes females more sensitive to Francisella tularensis infection
Authors: Spencer Charles T , Sanchez Guillen Michelle A
Published: January 2023

The intensity of the inflammatory response differs between males and females, with females having a stronger basal inflammatory response compared with males. This leads to differences in sensitivity to autoimmune and infectious diseases. Males tend to be more susceptible to infectious diseases whereas females tend to be more susceptible to autoimmune diseases. However, certain infectious diseases cause disease by over stimulating an inflammatory response resulting in dysregulation of cytokine production and generation of a cytokine storm. We hypothesized that the elevated basal inflammatory state of females would make them more susceptible to inflammatory infectious diseases such as Francisella tularensis. Indeed, survival and immunological studies demonstrated that females had a stronger innate immune response and were more likely to succumb to tularemic-disease compared with males. Importantly, this dichotomy was independent of hormonal signaling. We traced the source of this elevated inflammatory response to alterations in the M1 and M2 macrophage activation profile. In females, M1 macrophages were activated earlier than in males in response to inoculation. These M1 macrophages from female mice showed a stronger activation both in vivo and in vitro cultures. This was consistent in both mouse and human systems. Our results demonstrate that females possess a higher abundance of M1 macrophages and that these M1 macrophages are more inflammatory in response to F. tularensis infection. This suggests that the consequence of the dichotomy between the male and female inflammatory response is dependent upon the mechanism of disease triggered by infectious diseases.

Supported by grants from the NIH (SC1 GM122699-01A1) and NSF (MRI 1626587).
To Sir with Love! Students Feedback from an Immunology Course.
Authors: Eshetie Kirubel , Ngo Sylvie , Hailu Hailu , Desta Negatu , Fazal Nadeem
Published: January 2023

Students are obligated to write reviews and evaluate both their courses as well as their teachers at the end of the semester in anonymous surveys. The response rates from students have been continuously on decline as reported by third party evaluation companies. This has resulted in both failure of the purpose of evaluations as well as validity and statistical significance of these responses. We provided a non-binding, open and forum-like tool to get the perceptions and trends from Chicago State University College of Pharmacy students. Anonymous survey was sent using google form at the end of an Immunology course being offered to year-2 pharmacy students. Data from 29 respondents were collected and most significant results are summarized. When asked about their learning style; 45% loved traditional PowerPoint presentations as compared to 7% recorded lectures, 10% made their own notes, 3% were small group learners, while 31% solely relied on studying practice tests. When asked about the use of YouTube; 62% preferred random searches while 39% found Khan Academy videos as very useful. When asked about major barrier in learning Immunology; 38% reported test anxiety, 14% personal issues, 10% overwhelmed with curriculum, and 7% cited lack of motivation. In response to assessment questions, 92% students suggested that Instructors should not have more than 60 questions in 2-hour exam. Regarding exam question types, 90% preferred MCQs, 7% Essay, 14% Case-based, and 31% True/False. Following a 16-week, 2-credit course of Immunology, 54% got motivated to do research in Immunology. When asked about overall sentiments after taking a first course in Immunology. The unanimous response was that they all felt as if they were taking a course in a foreign language.
Antibody ‘Hotspots’ induce antibody-dependent cell-mediated cytotoxicity against SARS-CoV-2 spike-expressing lung fibroblasts
Authors: Holder Kayla , Hatfield Keeley , Ings Danielle , Fifield Katie , Harnum Debbie , Grant Michael
Published: January 2023

Since COVID-19 emerged in 2019, neutralizing antibody (Ab) responses have dominated as surrogate measures of protection against infection and severe illness. The mostly self-limiting course of SARS-CoV-2 infection suggests the innate immune system, including natural killer (NK) cells, plays a role in its control. Therefore, we investigated SARS-CoV-2-specific antibody-dependent cell-mediated cytotoxicity (ADCC) and if Abs against Wuhan and early SARS-CoV-2 variants activate ADCC against emergent highly transmissible variants. ADCC was evaluated by measuring NK cell cytotoxicity (51Cr) against plasma (1:1000)-sensitized MRC-5 cells stably transduced to express Wuhan or Delta spike. ADCC mediated by plasma from double-vaccinated and convalescent adults was generally low (range 1.2 – 24.9%; median 7.2% (IQR 5.2 – 10.6%), and plasma from those with previous natural infection mediated significant ADCC (> 10% lysis) to the same extent as plasma from fully vaccinated individuals (13/34 vs 11/44). Hybrid immunity imparted robust ADCC (27.9 ± 10.8% target lysis) in 32/34 individuals tested and levels of ADCC significantly correlated with IgG3 responses (P = 0.0093) against spike stalk (S2) domain. A peptide scan identified four distinct ‘hotspot’ regions associated with robust ADCC in individuals with reactive Abs. Overall, a minor decline in ADCC against Delta variant spike-expressing relative to Wuhan targets occurred in all groups; however, individuals with Abs against the region containing D614G mediated greater ADCC against Delta variant than Wuhan targets. Determining which Abs provide robust and broad ADCC can inform future COVID-19 prevention and treatment strategies and predict ADCC responses for emergent variants.

Supported by grants from CIHR through the COVID Immunity Task Force (VR1-173202)
Neutrophil NOX2 is Required to Control Experimental Urinary Tract Infection
Authors: Cotzomi-Ortega Israel , Han Rachel , Cortado Hanna , Li Birong , Becknell Brian , de Dios Ruiz-Rosado Juan
Published: January 2023

Urinary tract infections (UTIs) affect 150 million people worldwide. Uropathogenic Escherichia coli (UPEC) is the primary pathogen causing over 80% of UTIs. Systemic antibiotic treatment is the only available therapy for UTIs. Thus, novel non-antibiotic treatments for UTI are necessary. The NADPH oxidase complex 2 (NOX2) has an essential role in the antimicrobial response against many bacterial and fungal infections, but its role during UTIs remains unknown.

Objective

To study the role of NOX2 in bacterial killing during experimental UTI.

Methods

Female wild-type (WT) C57BL/6 mice, NOX2 knockout (KO) mice, and WT mice treated with a NOX2 inhibitor were transurethrally infected with UPEC (CFT073 strain). NOX2 protein expression was analyzed by Western blotting. Cellular sources of NOX2 were identified by immunofluorescence. Neutrophils were isolated from murine bone marrow. Human neutrophils were purified from peripheral blood of healthy donors. Purified neutrophils were infected with UPEC in vitro. UPEC burden was assessed by enumerating colony forming units (CFU). Reactive oxygen species (ROS) production was quantified using the Amplex™ Red kit.

Results

NOX2 KO mice, or mice treated with NOX2 inhibitor, were more susceptible to experimental UTI compared to WT mice. Immunofluorescence microscopy localized NOX2 primarily in infiltrating neutrophils. Neutrophils from NOX2 KO mice exhibited impaired extracellular ROS production and increased intracellular bacteria compared to WT controls, after challenged with UPEC in vitro. Similar effects occurred in human and murine neutrophils following chemical inhibition of NOX2 in vitro.

Conclusion

Neutrophil NOX2 serves a critical antimicrobial role during experimental UTI.

Supported by grant from NIH (K01 DK128379-01)
A novel function for the CoQ10 biosynthetic complex in anti-pneumococcal macrophage function
Authors: Walker Emma C , Todd Elizabeth , Ramani Rashmi , Anaya Edgar , Javati Sarah , Matlam John-Paul , Pomat William , Morley S Celeste
Published: January 2023

Despite the recent introduction of pneumococcal polysaccharide and conjugate vaccines, Streptococcus pneumoniae infection remains a leading cause of illness and death worldwide. In particular, infants in Papua New Guinea are at increased risk of severe pneumococcal pneumonia compared to infants in similar countries. We sought to determine if a novel genetic variant could explain this increased susceptibility. Whole exome sequencing revealed a single nucleotide variant (D308Y) in the gene encoding COQ6 (COQ6DY), a monooxygenase required for CoQ10 biosynthesis. We have utilized both a Saccharomyces cerevisiae model and a mouse model of COQ6DY to show that despite adequate production of CoQ10, this variant directly causes increased susceptibility to S. pneumoniae. This variant represents a previously unknown function of the CoQ10 biosynthetic complex that does not exert its effects through CoQ10 deficiency but rather through alterations of mitochondrial function and metabolism. These mitochondrial deficits in COQ6DY macrophages are sufficient to abrogate macrophage killing of S. pneumoniae and alter the coordination of the downstream immune response. In conclusion, we have identified a novel susceptibility allele to S. pneumoniae infection that exerts its effects via alterations in macrophage mitochondrial function.

Supported by NIH (R21 AI142723), SLCH Children’s Discovery Institute (PD-II-2018-742)
Examining the immune regulatory function of NLRP12 through novel protein interactions
Authors: Rippe Catherine A
Published: January 2023

NOD-like receptors (NLRs) are intracellular proteins that play an important role in the regulation of the innate immune response to pathogens. Since being identified, various functions for NLRP12 have been suggested. It has been shown to negatively regulate the inflammatory response by inhibition of the NF-kB signaling pathway and to exacerbate inflammation through the formation of a multi-protein complex called an inflammasome. Thus, the mechanisms by which this protein functions largely remain unclear. In this study, we examined the interaction between Cul3, a protein involved in protein ubiquitination, and NLRP12 to elucidate possible mechanisms by which it functions and/or is activated.

Supported by a grant from the Missouri State University Graduate College
Neutrophilic inflammation reprograms the bone marrow to impair T cell immunity during Tuberculosis
Authors: Saqib Mohd , Das Shreya , Mishra Bibhuti B
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection induces persistent influx of neutrophils that precludes both CD4 and CD8 T cells from tuberculosis (TB) lesions in genetically susceptible mice. Depletion of neutrophils restores T cell number and ameliorated disease. High neutrophil to T lymphocyte ratio in the peripheral blood of pulmonary TB (PTB) patients correlate with sputum smear positivity and disease severity. These observations led us to hypothesize that infiltration of neutrophils negatively affect the normally protective T cell response in TB lesions. By employing various mouse strains that present neutrophil-rich lung lesions, we demonstrated that instead of serving as canonical myeloid derived suppressor cells (MDSCs), neutrophils produce granulocyte colony stimulating factor (CSF3/G-CSF) to indirectly regulate T cell numbers in TB lesions. Neutrophil-derived G-CSF reprograms the bone marrow towards a granulocyte-biased hematopoiesis at the expense of lymphocyte progenitors. Transcriptome profiling of the isolated neutrophils from the lung, spleen and bone marrow revealed a type I interferon (IFN-I) response signature in these neutrophils. Hematopoietic cell-intrinsic IFN-I signaling (IFNAR1) is both necessary and sufficient to promote the pathologic granulopoiesis generating Mtb-permissive neutrophils while suppressing lymphocyte progenitors (CLPs) production. Finally, blockade of IFNAR1-signaling ameliorates disease pathology by preserving the hematopoietic equilibrium. Collectively, our work uncovers a mechanism by which type I IFN response in neutrophils along with G-CSF perturbs hematopoiesis in the bone marrow and negatively impacts T cell immunity in TB lesions.
GSDMD deficiency drives immunopathology in cutaneous leishmaniasis
Authors: Go Christina K , Grayczyk James , Brodsky Igor E , Scott Phillip A
Published: January 2023

Cutaneous leishmaniasis is a neglected tropical disease that results in severe, chronic lesions that are frequently resistant to parasite-directed therapy. Studies in patients and murine models indicate that NLRP-3 dependent IL-1β release is associated with more severe disease and treatment failure. One mechanism leading to IL-1β release as well as pyroptosis is the formation of pores by gasdermin D (GSDMD). To determine if GSDMD might contribute to IL-1β release and subsequent increased disease, we infected GSDMD knockout mice and wild-type C57BL/6 (WT) mice with Leishmania major and followed the course of infection. Contrary to our expectations, GSDMD KO mice infected mice developed more severe lesions compared with WT mice, but without a change in parasite burden. Because inflammasome activation without pyroptosis has been reported to lead to prolonged IL-1β release from hyperactivated dendritic cells, we tested whether blocking IL-1β signaling would prevent severe pathology in GSDMD KO mice. However, GSDMD KO mice treated with anti-IL-1β developed pathology similar to the untreated GSDMD KO mice, indicating that IL-1β was not mediating the increased pathology. Therefore, we next compared the immune responses in WT and GSDMD KO mice. The only significant difference found was an increase in the production of IFN-g, and our working hypothesis is that excess IFN-g may contribute to the disease severity. Why the absence of GSDMD leads to increased disease remains unclear, but previously it was found that the loss of GSDMD can have consequences beyond IL-1β release and pyroptosis. Current studies are focused on defining the mechanism(s) leading to increased disease in GSDMD KO mice.

Supported by grants from NIH (5R01AI125265-05, 1R01AI150606-01, T32AR7465).
Molecular basis for NEK7-mediated NLRP3 inflammasome activation
Authors: He Yuan , Jeltema Devon , Wang Jihong , Cai Juan , Kelley Nathan , Yang Zhe
Published: January 2023

The NLRP3 inflammasome is a critical component of the innate immune system that is activated by microbial infections and cellular stress signals. The molecular mechanism of NLRP3 inflammasome activation remains not fully understood. As an NLRP3-interacting partner, NEK7 has emerged as a critical mediator for NLRP3 inflammasome activation. In contrast to NEK7, NEK6, the closely related member of the NEK family, does not support NLRP3 inflammasome activation. Here, we show that the NEK7 catalytic domain, which shares a high sequence identity with the counterpart of NEK6, mediates its interaction with NLRP3 and inflammasome activation. Within their catalytic domains, a single amino acid residue at a corresponding position (R121NEK7, Q132NEK6) differentiates their function in NLRP3 inflammasome activation. Surprisingly, the substitution of the glutamine residue to arginine residue at position 132 confers NEK6 the ability of NLRP3 binding and inflammasome activation. In addition, NEK7 is reported to interact with the LRR domain of NLRP3. We show that the LRR domain self-associates independently of NEK7 and promotes inflammasome assembly and activation in macrophages at the physiological condition.

Supported by NIH R01AI148544
A TLR4-independent critical role for CD14 in intracellular LPS sensing
Authors: Vasudevan Swathy Ollikara , Russo Ashley , Kumari Puja , Vanaja Sivapriya Kailasan , Rathinam Vijay
Published: January 2023

Intracellular sensing of LPS is crucial for antimicrobial defense and sepsis. Noncanonical inflammasome sensing of cytosolic LPS via caspase-4/11 triggers gasdermin-D mediated pyroptosis and the release of cytokines and alarmins. During infection, LPS associated with bacteria or bacterial outer membrane vesicles gains access to the cytosol. However, free LPS can access the cytosol in vivo. The mechanism by which free LPS gains access to the cytosol and activate the caspase-11-noncanonical inflammasome in vivo mostly remains elusive. CD14, a GPI-anchored glycoprotein, binds and transfers LPS to the TLR4-MD2 complex. While the role of CD14 in TLR4-mediated host responses to extracellular LPS is clear, its role in the noncanonical inflammasome sensing of cytosolic LPS is poorly defined. Here, we found that cytosolic LPS-induced GSDMD cleavage, IL-1β, IL-18 and DAMP release, pyroptosis and lethality were remarkably impaired in CD14-deficient mice, but not in TLR4-deficient mice. By using Cd14−/− and Casp11−/− mice strains on a Tlr4−/− background, we have decoupled CD14’s role in caspase-11 activation from its known function in TLR4 signaling. Mechanistically, CD14 mediates caspase-11 activation by facilitating the internalization of LPS in a TLR4-independent manner. Overall, our findings highlight a new TLR4-independent role of CD14 in noncanonical inflammasome activation by intracellular LPS in vivo.
Secreted IgM modulates the pool of IL-10 producing B Cells
Authors: McGettigan Shannon E , Aira Lazaro E , Kumar Gaurav , Baumgarth Nicole , Debes Gudrun F
Published: January 2023

Regulatory B cells (Bregs) are critical for maintaining immune tolerance, and alterations in Breg numbers has detrimental effects in inflammatory diseases, infection, and cancer. Bregs are found among most B cell subsets and act primarily by producing IL-10. Despite their importance, the signals that control Breg differentiation and maintenance are not well defined. Here, we demonstrate that mice incapable of secreting IgM (sIgM−/−) have drastically increased IL-10+ Bregs in lymphoid organs compared to wildtype (WT) mice. These IL-10+ Bregs are polyclonal and increased among all major B cell subsets. While newborn WT mice (<24 hr of age) harbor detectable serum IgM, IL-10+ B cell expansion occurs early after birth in both WT and sIgM−/− mice. However, IL-10+ Bregs contract only in WT but not in sIgM−/− mice, which is in parallel to rising levels of serum IgM in WT mice. SIgM−/− B cells that develop in the presence of circulating IgM express IL-10 similar to that of WT B cells within the same mouse indicating that the expansions in IL-10+ B cell populations are not due to B cell-intrinsic effects based on the inability to secrete IgM, but rather the presence of sIgM. Mice that lack B cell-expressed high-affinity IgM receptor (FcμR) mirror the IL-10 phenotype in their B-1 and marginal zone B cells, but not follicular B cells, suggesting that secreted IgM can act as a negative regulator of IL-10 expression in B cell subsets via binding to surface-expressed FcμR, and potentially other IgM binding receptors. Our data indicate that sIgM controls IL-10 competence in B cells via IgM binding receptors thereby revealing potential novel avenues for altering IL-10+ Breg induction in disease settings. Funding: T32AI134646, R01AR067751, R01AI127389, R01AI148652.

Supported by grants from NIH (T32AI134646, R01AR067751, R01AI127389, R01AI148652)
The CD40/CD40 ligand system in linking acute neuroinflammation with chronic progressive demyelination
Authors: Sarma Jayasri Das , Saadi Fareeha , Chakravarty Debanjana , Kamble Mithila , Kumar Saurav , Shindler Kenneth S
Published: January 2023

CD4+ T cells play critical roles in mediating adaptive immunity and developing protective host immunity by eliminating infectious viruses. CD40 Ligand expressed on CD4+ T cells known to interact with its cognate receptor CD40 expressed on monocyte/macrophage/microglia to induce several immune functions during inflammation and host immunity. Our study aimed to delineate a potential CD4+ T cell-microglia nexus at the cellular and molecular level using CD4−/− and CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to murine-coronavirus-MHV-A59/RSA59 infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase of neuroinflammation. CD40L deficiency significantly impaired the priming, expansion, and activation of lymphoid cells in the deep cervical lymph nodes. Additionally, both CD4−/− and CD40L−/− mice presented with severe chronic phase demyelination and axonal pathology in the brain and spinal cord which was correlated with prolonged virus persistence. Results from this study provide exciting information about a critical role of CD40L in mediating RSA59 induced activation of CD4+ T cells in the CLN and their consequent infiltration into CNS in addition to affecting the numbers and activation of monocyte/macrophage/microglia during the acute-adaptive transition phase. CD40-CD40L interaction in linking the innate-acute and chronic-adaptive immune responses may suggest a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to autoimmune suppression, which holds only for autoimmune mechanisms of inflammatory demyelination.

This work was supported by a Department of Biotechnology, India, research grant (BT/PR 20922/MED/122/37/2016). We thank the Council of Scientific and Industrial Research (CSIR) India for providing fellowships to F.S. the Ministry of Human Resource Development (MHRD), India for providing fellowship to D.C; and the University Grants Commission (UGC), India, for providing fellowship to M.K.
Soluble and membrane-bound isoforms of fractalkine differentially regulate microglia activation and vascular damage in the diabetic retina
Authors: Rodriguez Derek , Church Kaira A , Pietramale Alicia N , Cardona Sandra M , Vanegas Difernando Vanegas , Nash Kevin R , Cardona Astrid E
Published: January 2023

Diabetic retinopathy (DR) is the leading cause of blindness worldwide due to neuronal loss, microgliosis, and vascular damage. Inflammation caused by microglia exacerbates retinal damage, resulting in glial and neuronal cell dysfunction. Microglial-neuronal crosstalk mediated by CX3CR1/FKN signaling regulates microglial responses. CX3CR1 polymorphic variants present in 25% of the population decrease FKN binding affinity to CX3CR1, and their role in microglia function in the retina during diabetes is unknown. FKN exists as a membrane-bound (mFKN) adhesion molecule. When cleaved by proteases, the soluble (sFKN) form exerts chemoattractant properties. Previous studies show that disruption of CX3CR1/FKN signaling worsens retinal pathology and can be rescued by administration of recombinant FKN. Mechanisms by which mFKN and sFKN regulate retinal function are still unknown. We hypothesize that during diabetes, the over-expression of sFKN using recombinant adeno-associated viruses (rAAVs) will prevent vascular and neuronal damage, and improve visual function. To test this hypothesis, rAAVs expressing mFKN or sFKN were delivered to FKN-KO mouse retinas. We validated the expression of FKN levels in retinal protein extracts in mice that received rAAVs expressing mFKN or sFKN. Our findings revealed that rAAV-sFKN minimizes microglial activation, reduces fibrinogen deposition, and rescues neuronal loss, compared to rAAV-mFKN. rAAV-sFKN retinas of diabetic and non-diabetic mice improved visual function in a two-choice visual discrimination task. FKN appears to be neuroprotective, serving as an alternative therapeutic approach to minimize retinal pathology and improve visual function.

Supported by NIH/NEI RO1 EY029913
The role of ionotropic AMPA receptors in T cell tolerance
Authors: Mitchell-Flack Marisa , Zheng Ying , Goldschmidt Hana , Rajkovich Kacey , Higgins Makenzie , Liu Bian , Huganir Richard , Yu Hong , Pardoll Drew
Published: January 2023

The ability of T cells to mount effective immune responses to pathogens and cancer while remaining tolerant to self-tissue is critical for maintaining immune homeostasis. Previously, our group identified neuritin, a neuronal protein, as a highly differentially expressed gene in anergic and regulatory T cells. Furthermore, our results indicate neuritin deficient mice exhibit enhanced autoimmunity due, in part, to dysfunctional regulatory T cells. Neuritin was recently identified as an accessory component of the ionotropic AMPA receptor (AMPAR) complex in neurons. AMPAR complexes are typically found on the post-synaptic neuronal cell and mediate glutamate dependent cation flux. Given our previous findings involving neuritin in autoimmunity and this newly identified interaction between neuritin and AMPAR, we sought to evaluate whether there is a role for the AMPAR in T cell tolerance. Here, we show that the AMPAR is expressed in CD4+ T cells and that the specific deletion of the AMPAR in T cells (AMPAR KO) leads to significantly reduced disease pathogenesis in an experimental autoimmune encephalomyelitis (EAE) model. The spinal cord inflammatory immune infiltrates from AMPAR KO mice are markedly reduced in cell number, proliferative state, and expression of proinflammatory cytokines compared to wild type (WT) mice. In addition, we have observed an increase in the CD4+FoxP3+ regulatory cell population among spinal cord infiltrates from AMPAR KO EAE mice. Consistent with the EAE model, AMPAR KO T cells also exhibit increased induced regulatory T cell development during in vitro differentiation. Taken together our results support a previously unappreciated role for ionotropic AMPA receptors in regulating T cell tolerance.
Microglia depletion elicits neuroprotective effects to alleviate vascular damage and neuronal cell loss in the diabetic retina
Authors: Church Kaira A , Rodriguez Derek , Lopez-Gutierrez Irene , Vanegas Difernando Vanegas , Cardona Sandra M , Cardonaa Astrid E
Published: January 2023

Diabetic retinopathy (DR), an incurable eye disease, is a common complication of diabetes mellitus and the leading cause of blindness amongst working age adults. Prolonged high glucose levels damage retinal blood vessels leading to hemorrhages, ischemia and ultimately vision loss. Microglia, the resident macrophages of the central nervous system, are rapidly activated and respond to transient hyperglycemia, and reset the homeostatic threshold of the retina. As hyperglycemia persists, capillary damage occurs resulting in serum proteins and DAMPs from the periphery leaking into the retina perpetuating microgliosis, pro-inflammatory cytokine production and vascular damage that correlates with neuronal loss. Therefore, we transiently depleted microglia to determine the mechanism by which resident retinal macrophages regulate neuronal and vascular damage at early stages of diabetes. We hypothesize that transient depletion of CX3CR1CreER:R26iDTR microglia during the course of disease when microglia receive the early-stage environmental cues of retinal inflammation due to hyperglycemia will induce neuroprotective cues. Utilizing a genetic model to express diphtheria toxin receptor under the tamoxifen inducible CX3CR1-promoter, microglia become susceptible to the effects of diphtheria toxin. Our findings revealed that transient depletion of microglia induced neuroprotective cues to upregulate TUJ1+ neurons and ameliorate vascular damage in the diabetic retina. In conclusion these studies suggest that early transient microglia depletion is neuroprotective by inducing the proliferation of a homeostatic microglia cell population that induce axonal regeneration of TUJ1+ neurons and vascular repair.

Supported by grants from NIH (R01EY029913) and NIH RISE Grant (GM061655).
Chimeric antigen receptors on Regulatory T cells as a treatment strategy in autoimmune diseases
Authors: Dasari Harika
Published: January 2023

Research question

Can CAR-MOG Tregs help curb the neuroinflammation in a MS mouse model?

Objectives

Methods

To test this out, we sorted Tregs from human peripheral blood or murine spleens using T cell enrichment followed by flow-cytometric sorting and amplified using Dynabeads (human CD3/CD28 activator) for Tregs proliferation and stained for Treg markers to ascertain the purity of isolated Tregs. We then constructed a second-generation CAR against MOG monoclonal antibody (8.18 C5) and transduce Tregs and Tconvs to test the transduction efficacy and suppressive function of CAR-Tregs. To test the killing function of CAR-MOG-Tconv and compared this to that of CAR MOG Tregs, we set up a cytotoxicity assay against a cell line modified to express MOG. Additionally we performed suppression assays with control Tregs and CAR transduced Tregs to determine if CAR transduction affects the immunosuppressive capacity of Tregs.

Results

CAR Tconv and CAR Tregs show specificity against K562 cell line modified to express MOG, CAR Tregs have a lower killing capacity compared to CAR Tconv. CAR Tregs do not lose their immunosuppressive function and are comparable to the untransduced Tregs. CAR Tregs upon co-culture with Target cell line (K562 MOG) express activation markers and increased expression of cytokines such as IL-10, and TGF beta.

Supported by Canada Graduate Scholarships Doctoral Award (CGS D)
TGF-β signaling drives phenotypic and functional characteristics of human mast cells within epithelium
Authors: Derakhshan Tahereh Mina , Hollers Eleanor , Buchheit Kathleen , Laidlaw Tanya M , Boyce Joshua A , Dwyer Daniel
Published: January 2023

Mast cells (MCs) are key effector cells in the development of type 2 inflammation, such as asthma and nasal polyposis. Intraepithelial MCs are phenotypically distinct from those found within the subepithelium and preferentially expand during mucosal inflammation, yet the mechanism driving their development and their relative contribution to inflammation is not clear. We previously found that TGF-β directed the murine intraepithelial airway MC phenotype. Here, we investigate whether TGF-β has a similar effect on human MCs. Using single cell RNA-sequencing of primary nasal polyp MCs and bulk sequencing of in vitro peripheral blood-derived MCs (PB-MCs) stimulated with TGF-β, we identified a shared signature of TGF-β within human intraepithelial MCs. This included downregulation of subepithelial MC-associated granule components (NDST2, CMA1, and CTSG), differential regulation of lipid mediator biosynthetic enzymes (PTGS1, HPGDS), and upregulation of transcription factors (SKIL, FOXP1). TGF-β suppressed incorporation of the proteases chymase and cathepsin G into PB-MC granules in vitro as part of a broader intraepithelial MC phenotype, allowing direct assessment of the differential role of MC subsets during inflammation. Through this approach, we found that TGF-β modifies MC production of inflammatory mediators in response to stimulation, enhancing MC production of the lipid mediators, PGD2 and CysLTs, in response to IgE-crosslinking; selectively decreasing IL-13, TNF-α, and IL-6; and enhancing chemokine production (IL-8 and MIP-1β). Thus, we identify a central role for TGF-β signaling in driving the human intraepithelial MC phenotype and re-shaping their response to activation.

Supported by NIH (K22 AI146281, U19AI070535)
Leveraging iNKT cells to identify signals driving B cell memory commitment
Authors: Parthasarathy Raksha , Yates Jennifer , Hagglof Thomas , Dudley Elizabeth A , Leadbetter Elizabth
Published: January 2023

Intense scrutiny of the process of memory B cells (MBC) over the last decade has improved our understanding of MBC development especially for vaccines against viruses capable of rapid diversification like SARS-CoV-2. Most memory responses develop in germinal centers (GC), but the constellation of specific signals which commit B cells to a memory fate is poorly understood. Identification of discrete T and B cell factors leading to MBC commitment is hindered by a lack of murine models with essential controls. We report a unique system harnessing iNKT cell help for B cells to create two conditions, both of which expand NP+ B cells in a class-switched primary response, but only one of which produces a MBC response. In the first case, NP+ B cells receive exclusive cognate iNKT cells upon immunization with the Nitrophenyl-haptenated iNKT cell agonist alphaGalactosylCeramide (NP-aGC) leading to an expansion of NP+ GC B cells, but not MBCs. In contrast, NP+ B cells helped by conventional T cells adjuvanted by iNKT cell activation by vaccinating with haptenated protein (NP-KLH) plus aGC drives a conventional GC B cell expansion and development of NP+ MBCs. Interestingly, cognate antigen which does not induce B memory also elicits fewer pre-memory B cells, compared to non-cognate antigen. These different immunization regimens allow a unique comparison of activated B cells recognizing identical epitopes and producing similar primary humoral responses but with contrasting memory outcomes. Importantly, this also controls for the GC environment which is unaccounted for in other model systems. Examination of these B cell populations will better define the transcriptional landscape of MBC and pinpoint key signals which dictate MBC fate.

Supported by AAI Careers in Immunology Fellowship 2021
Mechanism of Tim-3 regulation of CD8+ T cell function
Authors: Manandhar Priyanka , Landy Emily Elizabeth , Murter Benjamin , Banerjee Hridesh , Workman Andrea , Bosco Isabella , Kane Lawrence P
Published: January 2023

Tim-3 or transmembrane immunoglobulin and mucin domain-3 is a type I membrane protein expressed by various immune cell types, and has been shown to have a co-stimulatory role in T cells through the PI3K pathway. Tim-3 comprises extracellular, transmembrane and intracellular domains, the latter of which contains five tyrosine residues. We and others have shown that these tyrosines participate in TCR-mediated signaling pathways. Using LCMV infection, we also found that Tim-3 expression influences the formation of short-lived effector and memory precursor CD8+ T cells. We hypothesize that Tim-3 signaling through the tyrosine residues in its cytoplasmic domain modulates CD8+ T cell activation and differentiation. To test this hypothesis, we have used LCMV-specific TCR transgenic (P14) mice crossed to truncated version of Tim-3 and CD8+ conditional deletion of Tim-3. CD8+ T cell activation and memory were analyzed by flow cytometry after infection with LCMV Armstrong to induce an acute infection. The effects of Tim-3 on CD8+ T cells were found to be most prominent at the effector stage of infection and RNA sequencing has shown that loss of Tim-3 signaling promotes acquisition of a memory-like phenotype. Transcription factors T-bet and Blimp-1 were found to be lower in Tim-3 KO CD8+ T cells, while Tcf-1 maintained at higher levels in the absence of Tim-3 signaling. Cytokine production by effector CD8+ T cells was also found to be lower when Tim-3 was absent. Thus, we conclude that Tim-3 signaling via its cytoplasmic tyrosine residues plays a role in CD8+ T cells in an acute infection. These results may lead to a better understanding of CD8+ T cell activation and memory development in different settings.

Supported by R01 AI138504-01A1
Enhanced cytotoxic CD8+ T cell (CTL) antigen recognition by LCK activation
Authors: Zheng Yating , Piper Taylor L , Courtney Adam H
Published: January 2023

The anti-tumor response of cytotoxic T cells requires detection of tumor-derived antigenic peptide-MHC complex (pMHC) by the T cell antigen receptor (TCR). Strategies that boost TCR signaling caused by weak pMHC could augment the recognition of poorly immunogenic tumors by T cells. Because LCK kinase phosphorylates the TCR complex and the recruited ZAP-70 kinase, increased LCK activity has been shown to increase TCR signaling in naïve T cells. Our previous study demonstrated that a small molecule inhibitor of CSK kinase, a negative regulator of LCK, increased LCK kinase activity and sensitized naïve T cells such that they became activated by weak antigens ex vivo. Our goal was to determine whether CD8+ cytotoxic T cells (CTL) could also be sensitized to detect weak antigens, and evaluate whether their capacity to recognize and kill cancer cells could be augmented. Because the Nur77-GFP reporter system responds to TCR-mediated signaling, we used it to determine that LCK activation sensitized OT-1 TCR transgenic CTLs to detect weak (low affinity) antigens displayed by splenocytes. We also observed increased phosphorylation of ZAP-70 and other signaling mediators upon TCR stimulation. We will next evaluate whether CTL-mediated effector functions can be enhanced by monitoring killing of MC38 cancer cells that display a weak antigen. Our findings will inform whether LCK kinase can be targeted to improve the recognition of cancer by T cells.
Tumor suppressor p53 controls thymic NKT17 cell development
Authors: Celli Sofia , Watanabe Masashi , Hodes Richard J
Published: January 2023

Natural killer T (NKT) cells are a lineage of innate-like lymphocytes that develop in the thymus, where they acquire effector phenotypes and differentiate into subsets, NKT1, NKT2, or NKT17. NKT2 cells express the highest PLZF levels; NKT1s depend on IL-15 signaling and express T-bet; and NKT17s depend on TGFβ and express RORγt. Although several factors distinguishing these subsets have been identified, more remains to be elucidated; in exploring such factors, we found that the tumor suppressor p53 plays a role in selectively regulating thymic NKT17s. In mice with T cell-specific p53-deficiency, thymic NKT17s are expanded up to tenfold. Among thymocyte populations analyzed, wildtype thymic NKT17s express the highest levels of p53, and upregulate the highest levels of p53 in response to irradiation. We examined γH2AX as a marker of DNA double-strand breaks, and found that the p53-deficient NKT17 subset had the highest γH2AX expression among all analyzed thymocyte populations. These results indicated that NKT17s have selectively greater genomic instability than other thymocyte populations and suggest a role for p53 in maintaining the expansion of the thymic NKT17s, potentially in response to genomic instability in this population. We are currently investigating the functional status of NKT17 cells in p53-deficient mice, as well as seeking to assess how this significantly increased “type 17” population impacts the thymic niche.
Apolipoprotein E enhance survival of effector memory regulatory T lymphocytes by regulating caspase-dependent apoptosis and lipid metabolism.
Authors: Atehortua Laura Melissa , Street Scott , Davidson W Sean , D’Alessandro Angelo , Chougnet Claire
Published: January 2023

Mechanisms underlying the pleiotropic anti-inflammatory effect of HDL remain incompletely understood. Regulatory T cells (Tregs) are essential for maintaining immune homeostasis in cardiovascular diseases (CVD) and metabolic disorders. We recently show Tregs, particularly effector memory Tregs (emTregs), bind/internalize HDL, which promotes their survival by limiting caspase-dependent apoptosis. As HDL contain a complex mix of proteins and lipids, we investigated which components of HDL mediate this process and found HDL proteins were the most critical. Using reconstituted HDL mimic vesicles containing recombinant HDL proteins and POPC, we found that APOE, but none of the APOA proteins, significantly decreased caspase expression in emTregs similarly to total HDL. Neither APOE nor HDL had any effect on naïve Tregs. APOE-depleted HDL did not rescue emTregs, confirming the critical role of APOE. Because Tregs have distinct metabolic requirements, we next investigated the effect of HDL, APOE3-POPC and APOE-depleted HDL on lipid metabolism in emTregs. APOE3-POPC and HDL significantly increased the concentration of long chain (14:0 20:0) and polyunsaturated fatty acids (FA, 18:3 22:6) in emTregs. Our data also suggested the involvement of de novo FA synthesis, as APOE3-POPC and HDL increased the concentration of palmitate (16:0), the earlier precursor of this pathway. Further, treatment of Tregs with the fatty acid synthase inhibitor C75 abolished HDL survival effect. Our results show that HDL, specifically APOE, promote emTregs survival by regulating de novo FA synthesis and caspase-dependent apoptosis. HDL-APOE levels seem to be dysregulated in CVD and obesity, suggesting this mechanism may contribute to HDL protective role
Th1 and Th17 cells are critical for limiting the severity of Staphylococcus aureus craniotomy infection
Authors: Kak Gunjan , Horn Christopher M , Heim Cortney E , Kielian Tammy
Published: January 2023

A craniotomy is performed to access the brain for tumor resection, localization and resection of epileptogenic foci, and aneurysm clipping. Despite prophylaxis, infectious complications after craniotomy range from 1–3%, with approximately half caused by Staphylococcus aureus (S. aureus), which forms a biofilm on the bone flap that is recalcitrant to antibiotics. Using our novel mouse model of S. aureus craniotomy infection, Th1 and Th17 infiltrates were found to preferentially home to the brain but not the subcutaneous galea; however, their functional importance is unknown. RAG1 knockout (KO) mice displayed significant increases in bacterial burden in the brain, galea, and bone flap at days 3, 7, and 14 post-infection, which was negated following the adoptive transfer of either in vitro skewed Th1 or Th17 cells. Interestingly, in vitro skewed Th17 cells acquired Th1 characteristics upon migrating into the brain in vivo as revealed by the production of Th1-associated cytokines such as IFN-γ and TNF-α with minimal IL-17A. We established that the window of T cell protection was during acute infection, since delaying Th1 or Th17 adoptive transfer until day 7 post-infection was no longer able to attenuate S. aureus burden in RAG1 KO mice compared to T cell transfers at day −1 or 3 after infection that were effective. In vitro studies established that both Th1 and Th17 cells augmented microglial, macrophage, and neutrophil S. aureus bactericidal activity and IL-12p70 and CXCL10 production. Collectively, these findings highlight the importance of an early adaptive immune response for bacterial containment during S. aureus craniotomy infection by promoting the antimicrobial activity of infiltrating leukocytes and resident microglia.