Innate lymphoid cells (ILCs) are the innate counterpart to CD4+ T cells that are mediated by the same transcription factors and produce similar cytokines. ILCs are being investigated in many different disease states, but the field current lacks foundational information on ILC representation whether it be in tissues, between males and females, or in aging as these are all vital components in disease etiology and severity. Our descriptive study used flow cytometry to characterize ILCs compared to the entire CD45+ (e.g., lymphocyte) and lineage negative (e.g., ILC) compartments to understand their homeostatic balance and plasticity. Moreover, we defined ILC2 expression and subsets based on their cytokine production and created several mathematical models to elucidate the correlation of extra- and intra-cellular ILC2 markers from least to most complex. ILC studies would benefit from more unbiased, holistic experiments including RNA-seq and mass spectroscopy to further define ILCs in steady-state before adding more complex pathways like different disease states to enhance translational value and therapeutic targeting of these cells.

Background: Lactoferrin (LTF) is an essential, high-affinity iron-binding protein that is abundant in the secondary granules of neutrophils. After intracerebral hemorrhage (ICH), circulating blood neutrophils (PMNs) enter the ICH-afflicted brain and secrete LTF into the damaged tissue. This LTF can bind to iron and hemoglobin (key toxic components of the hematoma) thereby neutralizing toxicity of the hematoma. Here we study the role of LTF in the rodent brain after ICH. Methods and Results: In an autologous blood injection model of ICH in rats using RT-PCR and Western blotting, we measured the expression of LTF in ICH-affected brain. We found that LTF mRNA is virtually undetectable in the naïve rats’ brains and brains of rats at 3h to 7d after ICH. These findings suggest that LTF is not synthesized in the brain. LTF protein was not detectable in the naïve rat brain; however, the LTF protein is increased starting 3-6h post-ICH in the hematoma-affected hemispheres, reached maximum by 24-48h, and remained elevated for about 7d. Double immunofluorescence for LTF and RP-1 antigen (a biomarker for PMNs) shows that most LTF + -cells in the ICH-affected brains are PMNs. The locations of LTF + -cells in the brain were primarily peri-hematoma and the hematomas, which are loci of PMNs infiltration. Also, the temporal profile of LTF level and the number of PMNs in the brain were similar. We also detected a similar progression of LTF changes in the peripheral blood after ICH. Finally, in the ICH model in rats, administration of the recombinant LTF (rLTF) after the onset of ICH significantly reduced brain edema, oxidative damage, and neurological deficits, as assessed on day 3. Interestingly, in neuron-glial co-culture, after exposing these cells to red blood cells (RBCs; to simulate ICH-mediated injury), rLTF reduced the neurotoxic effect of RBCs and accelerated the removal of RBCs by microglia. This suggests that the cytoprotective effect of LTF and the modulation of microglia’s phenotype could underlie the beneficial role of LTF, with neutrophils being a primary source of LTF production. Conclusion: We propose that LTF delivered to the ICH-affected brain by infiltrating activated PMNS could be beneficial for hematoma detoxification, which may be a potential target for ICH therapy.

Microglia/macrophage (MΦ) are immune response cells with function in clearing cell debris and tissue repair after ischemic brain damage. Retinoid X receptor alpha (RXRα) is a pleiotropic transcription factor regulating cell differentiation, lipid/glucose metabolism and immune responses, including in MΦ. Studies from this lab suggest that the dimerization of RXRα with the peroxisome proliferator-activated receptor γ (PPARγ) to form a transcriptionally active structure plays critical roles in enhancing MΦ polarization toward the “healing” phenotype. All studies were performed applying scientific rigor. First, we showed that treatment of microglia in culture with selective agonist of the RXRα, bexarotene (BEX), promotes microglia’ polarization toward “beneficial” phenotype including enhancement of phagocytic functions toward engulfment of dead neurons. Next, we showed that BEX (5mg/kg, i.p.) given daily to rats after thromboembolic stroke reduced the infarct volume at d7. The neurological deficit was reduced by BEX at d7, but not at d3 after stroke. This suggest that the beneficial effects of BEX takes place during the sub-acute phase post-stroke. To further investigate the role of MΦ RXRα in ischemia, we generated macrophage-targeting RXRα knockout mice (MacRXRα -/- ) by crossing Lyz-Cre mice with RXRα flox/flox mice. We subjected MacRXRα -/- and RXRα flox/flox (control) mice to a 60 min MCA/CCA reversible occlusion. 24h later mice received BEX (5mg/kg once a day for 7 days, i.p.) or vehicle. We conducted behavioral evaluations for 28 days. We showed, in agreement with the rat data, that control mice receiving BEX showed significant improvement in long-term neurological recovery and had reduced brain atrophy volume at d28 after stroke. We also showed that beneficial effect of BEX is significantly reduced in MacRXRα -/- mice, as compared to control mice. Limitation: Since MacRXRα -/- mice may also have RXRα deficiency in neutrophils, we are now conducting parallel studies to evaluate the role of RXRα deficiency in neutrophils. Our study suggests that RXRα in the MΦ may play important role in coordinating the cleanup and repair during post-stroke recovery phase. Also, we showed that BEX has the uniquely long 24h therapeutic window.

Aging is a non-modifiable risk factor for stroke. Aging is accompanied by chronic low-grade inflammation and gut dysbiosis (a pathological imbalance of microbial organisms in the gut). Age-related gut dysbiosis exacerbates stroke outcomes and can be reversed by manipulation of the gut microbiota (GM) via fecal microbiota transplants (FMTs) from young animals, or “rejuvenation.” But, the mechanisms that mediate these effects are poorly understood. Dendritic cells (DCs) are potent antigen presenting cells and uniquely equipped to mediate the effects of dysbiosis. DCs constantly sample their environment to regulate the inflammatory response to antigens and tissue injury. In this study we investigated the role of intestinal DCs in mediating the detrimental effects of dysbiosis on stroke outcomes. We hypothesize that age-related dysbiosis exacerbates stroke outcomes by inducing an inflammatory and migratory phenotype in intestinal DCs. We studied four cohorts of C57Bl6 mice consisting of (1) naïve young (4mo), (2) naïve aged (22-26mo), (3) middle-aged (14mo) with FMT from aged donors, and (4) naïve young with 60-min middle cerebral artery occlusion (MCAO). Phenotyping of DCs by flow cytometry was performed. Results: In our MCAO cohort, we found a significant increase in activated DCs in the gut (1.4% vs. 7.6%, p = 0.051) but a decrease in frequency of activated DCs in the brain (8.4% vs. 3.9%, p = 0.042). In our FMT cohort, frequency of intestinal DCs was altered in a subset-specific manner after FMT from aged donors. Specifically, our data showed that the MHC-II expression by DC subsets with a migratory phenotype (CD11b + DCs) and resident DCs (CD103 + DCs) were significantly increased when middle-aged mice received FMT from aged donors (p < 0.05). In our naïve cohorts, we found a significant decrease of MHC-II surface expression in brain DCs (p = 0.044) and a significant increase in splenic DCs (p = 0.049) with aging. Conclusion: Our findings show that frequency and maturity state of DCs significantly differ with aging in a tissue- specific manner and can be influenced by manipulation of the gut microbiota. Our data also support the notion that intestinal DCs are involved in mediating the detrimental effects of age-related gut dysbiosis on stroke outcomes.