Brown Norway (BN) rats display an extremely low hypercapnic ventilatory response (HCVR) compared to other strains such as the Dahl S (SS) rat. Multiple pH‐sensitive potassium (K+) channels are expressed within multiple brainstem chemosensory nuclei and may contribute to CO2‐H+ detection/signaling in chemosensitive neurons. Herein we tested the hypothesis that there are selective reductions in the numbers of neurons expressing pH‐sensitive Kv1.4, Kir 2.3, and TASK‐1 K+ channels in the chemosensory nucleus of the solitary tract (NTS) but not in the pre‐Botzinger Complex (pre‐BötC), nucleus ambiguus (NA) or hypoglossal nucleus (XII) of BN but not SS rats. Medullary sections (25 µm) from male and female BN (n=14) and SS (n=15) rats were stained with cresyl violet or primary antibodies targeting KV1.4, Kir2.3, or TASK‐1 and the numbers of K+ channel‐immunoreactive (‐ir) neurons were quantified. There was little difference among female and male rats within each strain in all nuclei studied. However, within the NTS there were significantly fewer Kir2.3, (47.46% ± 1.83%; TASK‐1 57.46% ± 2.17%; and Kv1.4 64.11% ± 1.6% observed in BN rats as compared to SS rats. In contrast, the numbers of K+ channel‐ir neurons did not significantly (P< 0.05) differ within the NA and the hypoglossal nucleus, with some significant (P < 0.05) differences observed in the Medullary Raphe and the pre‐Botzinger complex. Fewer pH sensitive K+ channel‐immunoreactive neurons is evidence that at least partly explains the lower hypercapnic response in BN rats compared to that in SS rats.Grant Funding Source: Supported by the Department of Veterans Affairs and NIH HL25739

The ventilatory CO2 chemoreflex is inherently low in inbred Brown Norway (BN) rats compared with other strains, including inbred Dahl salt-sensitive (SS) rats. Since the brain stem expression of various pH-sensitive ion channels may be determinants of the CO2 chemoreflex, we tested the hypothesis that there would be fewer pH-sensitive K(+) channel-expressing cells in BN relative to SS rats within brain stem sites associated with respiratory chemoreception, such as the nucleus tractus solitarius (NTS), but not within the pre-Bötzinger complex region, nucleus ambiguus or the hypoglossal motor nucleus. Medullary sections (25 μm) from adult male and female BN and SS rats were stained with primary antibodies targeting TASK-1, Kv1.4, or Kir2.3 K(+) channels, and the total (Nissl-stained) and K(+) channel immunoreactive (-ir) cells counted. For both male and female rats, the numbers of K(+) channel-ir cells within the NTS were reduced in the BN compared with SS rats (P < 0.05), despite equal numbers of total NTS cells. In contrast, we found few differences in the numbers of K(+) channel-ir cells among the strains within the nucleus ambiguus, hypoglossal motor nucleus, or pre-Bötzinger complex regions in both male and female rats. However, there were no predicted functional mutations in each of the K(+) channels studied comparing genomic sequences among these strains. Thus we conclude that the relatively selective reductions in pH-sensitive K(+) channel-expressing cells in the NTS of male and female BN rats may contribute to their severely blunted ventilatory CO2 chemoreflex.

RATIONALE: Sendai virus (SeV) infection, similar to RSV, is characterized by early influx of polymorphonuclear neutrophils (PMN). We previously reported that with SeV infection two subsets of PMN are recruited to the lung, one of which (CD49d+) is critical for induction of the high affinity receptor for IgE on dendritic cells and subsequent post-viral atopic disease. Given that PMN have been reported to have the ability to inhibit T cell responses in the lymph node, we undertook this study to determine the ability of these PMN subsets to suppress T cell proliferation. METHODS: Bronchoalveolar lavage fluid (BALF) (>95% PMN) from C57BL6 mice on day 3 post-inoculation SeV was obtained. Based on CD49d expression, PMN subsets were separated by flow-sorting. CD8+ T cells from naïve OT-I/RAG1-/- mice were loaded with CFSE and cultured with OVA peptide, antigen-presenting cells, and varying concentrations of CD49d+ or CD49d- PMN. Cell proliferation was measured by flow cytometry after 72 hours using CFSE dilution of Thy1.2+ cells. RESULTS: CD49d- PMN significantly suppressed T cell proliferation at a PMN: T cell ratio of 1:30, whereas CD49d+ PMN failed to suppress T cell proliferation [Suppression of 40.4±1.8% (CD49d-PMN) versus 0.0±1.0% (CD49d+PMN), mean±SEM, p<0.001, n=4]. CONCLUSIONS: PMN subsets play different roles in a paramyxoviral infection, with CD49+ PMN inducing the atopic cascade and CD49d- PMN having a T cell suppressive role. Further studies will identify the relative importance of these subsets in the antiviral response.

RATIONALE: We previously demonstrated that a severe paramyxoviral (Sendai virus, SeV) respiratory infection in mice induces expression of the high-affinity IgE receptor (FcεRI) on lung conventional dendritic cells (cDC). FcεRI was expressed by 3 days post-inoculation (p.i.) in wild-type (WT) C57BL6 mice and this expression was necessary for the development of post-viral atopic disease. In humans, IgE is known to modulate FcεRI expression; therefore, the purpose of this study was to determine whether IgE plays a role in receptor induction.METHODS: WT and IgE deficient (IgE-/-) C57BL6 mice were inoculated intranasally (i.n.) with 2×105 pfu SeV. Three, 5, and 7 days p.i. mice were euthanized, lung cDCs were purified (>90%) by positive immunomagnetic bead selection, and FcεRI expression was assessed by flow cytometry.RESULTS: Induction of FcεRI in WT and IgE-/- mice was similar at day 3 p.i. with a fold increase in MFI of 2.65±0.97 (n=2) and 1.76±0.52 (n=3), respectively. At later time points there continued to be no difference between FcεRI expression on cDC from WT or IgE-/- mice (day 5 = 3.78±1.24 (n=2) versus 4.44 (n=1) and day 7 = 5.2±0.34 (n=5) versus 3.01±0.11 (n=5), WT and IgE-/- respectively).CONCLUSION: Unlike in mast cells and basophils where IgE tightly regulates FcεRI expression, our data suggest that IgE plays essentially no role in the paramyxoviral mediated induction or maintenance of FcεRI expression on murine lung cDC. RATIONALE: We previously demonstrated that a severe paramyxoviral (Sendai virus, SeV) respiratory infection in mice induces expression of the high-affinity IgE receptor (FcεRI) on lung conventional dendritic cells (cDC). FcεRI was expressed by 3 days post-inoculation (p.i.) in wild-type (WT) C57BL6 mice and this expression was necessary for the development of post-viral atopic disease. In humans, IgE is known to modulate FcεRI expression; therefore, the purpose of this study was to determine whether IgE plays a role in receptor induction. METHODS: WT and IgE deficient (IgE-/-) C57BL6 mice were inoculated intranasally (i.n.) with 2×105 pfu SeV. Three, 5, and 7 days p.i. mice were euthanized, lung cDCs were purified (>90%) by positive immunomagnetic bead selection, and FcεRI expression was assessed by flow cytometry. RESULTS: Induction of FcεRI in WT and IgE-/- mice was similar at day 3 p.i. with a fold increase in MFI of 2.65±0.97 (n=2) and 1.76±0.52 (n=3), respectively. At later time points there continued to be no difference between FcεRI expression on cDC from WT or IgE-/- mice (day 5 = 3.78±1.24 (n=2) versus 4.44 (n=1) and day 7 = 5.2±0.34 (n=5) versus 3.01±0.11 (n=5), WT and IgE-/- respectively). CONCLUSION: Unlike in mast cells and basophils where IgE tightly regulates FcεRI expression, our data suggest that IgE plays essentially no role in the paramyxoviral mediated induction or maintenance of FcεRI expression on murine lung cDC.

RATIONALE: We have shown that paramyxoviral respiratory infection induces expression of the high-affinity IgE receptor (FcεRI) on murine lung conventional dendritic cells (cDC). Cross-linking FcεRI results in CCL28 production, which recruits Th2 cells to the lung, and is associated with human and mouse models of asthma. We undertook this study to determine if other chemokines were produced upon cross-linking of cDC FcεRI. METHODS: C57BL6 mice were inoculated intranasally with 2x105 pfu Sendai virus. Five days post-inoculation mice were euthanized, lung cDCs were purified (>90%) by positive immunomagnetic bead selection, and FcεRI expression assessed by flow cytometry. cDCs were cultured with cross-linking anti-FcεRIα antibody (MAR-1) or control IgG for 6-30 h. After culture, mRNA was extracted from the cell pellets and analyzed for expression of CCL2 and CCL17 by real-time qRT-PCR, with data normalized to GAPDH mRNA levels. RESULTS: CCL2 expression was induced by FcεRI cross-linking at 6 and 30 hours (2.0±0.7 and 3.3±2 fold increase over IgG control, 6 and 30 hours, respectively, n=2). Similarly, CCL17 expression was also increased with FcεRI cross-linking (1.5±0.6 and 2.6±0.9 fold increase over IgG, 6 and 30 hours, respectively, n=2). CONCLUSIONS: Cross-linking FcεRI on lung cDC induces not only CCL28, but also message for CCL2 (a monocyte chemoattractant) and CCL17 (a Th2 cell chemoattractant associated with allergic disease). Whether these increases in message correlate with increased protein expression remains to be determined. Further studies will also explore the functional relevance of CCL2 and CCL17 in the paramyxoviral model of virus-induced atopic disease.

Ventilatory sensitivity to hypercapnia is greater in Dahl salt-sensitive (SS) rats than in Fawn Hooded hypertensive (FHH) and Brown Norway (BN) inbred rats. Since pH-sensitive potassium ion (K(+)) channels are postulated to contribute to the sensing and signaling of changes in CO(2)-H(+) in chemosensitive neurons, we tested the hypothesis that there are more pH-sensitive K(+) channel-immunoreactive (ir) neurons within the medullary raphé nuclei of the highly chemosensitive SS rats than in the other two strains. Medullary tissues from male and female BN, FHH, and SS rats were stained with cresyl violet or with antibodies targeting TASK-1, K(v)1.4, and Kir2.3 channels. K(+) channel-ir neurons were quantified and compared with the total neurons in the region. The total number of neurons in the medullary raphé 1) was greater in male FHH than the other male rats, 2) did not differ among the female rats, and 3) did not differ between sexes. The average number of K(+) channel-ir neurons per section was 30-60 neurons higher in the male SS than in the other rat strains. In contrast, for the females, the number of K(+) channel-ir neurons was greatest in the BN. We also found significant differences in the number of K(+) channel-ir neurons between sexes in SS (males > females) and BN (females > males) rats, but not the FHH strain. Our findings support the hypothesis for males but not for females, suggesting that both genetic background and sex are determinants of K(+) channel immunoreactivity of medullary raphé neurons, and that the expression of pH-sensitive K(+) channels in the medullary raphé does not correlate with the ventilatory sensitivity to hypercapnia.

Alzheimer’s disease, a common cause of dementia, is primarily tied to the formation of senile plaques consisting of deposition of the amyloid beta (Aβ) protein. Aβ has been implicated as a molecular link between oxidative stress and neuronal death in this disease state. Although the mechanism remains unclear, Aβ is known to induce free radical generation. In the present study, we chose to examine the enzyme cytochrome P450 (CYP) epoxygenase as a possible pathway for the generation of superoxide induced by Aβ protein. Using the superoxide detection probe hydroethidine and the recently developed fluorescence HPLC assay, we found that inhibition of CYP epoxygenase activity reduces the production of superoxide stimulated with the Aβ protein in rat brain astrocytes, and that evoked by alpha adrenergic receptor activation in tissues of Alzheimer’s patient brain. Antioxidant pretreatment attenuated the Aβ protein and alpha adrenergic stimulation induced generation of superoxide. These findings suggest that the CYP epoxygenase mediates Aβ and alpha adrenergic receptor activation stimulated superoxide production and could be a possible therapeutic target for lowering free radical level in Alzheimer’s disease.