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Bacterial infections following rhinovirus (RV), a common cold virus, are well documented, but pathogenic mechanisms are poorly understood. We developed animal and cell culture models to examine the effects of RV on subsequent infection with non-typeable Hemophilus influenzae (NTHi). We focused on NTHI-induced neutrophil chemoattractants expression that is essential for bacterial clearance. Mice infected with RV1B were superinfected with NTHi and lung bacterial density, chemokines and neutrophil counts determined. Human bronchial epithelial cells (BEAS-2B) or mouse alveolar macrophages (MH-S) were infected with RV and challenged with NHTi, TLR2 or TLR5 agonists. Chemokine levels were measured by ELISA and expression of IRAK-1, a component of MyD88-dependent TLR signaling, assessed by immunoblotting. While sham-infected mice cleared all NTHi from the lungs, RV-infected mice showed bacteria up to 72 h post-infection. However, animals in RV/NTHi cleared bacteria by day 7. Delayed bacterial clearance in RV/NTHi animals was associated with suppressed chemokine levels and neutrophil recruitment. RV-infected BEAS-2B and MH-S cells showed attenuated chemokine production after challenge with either NTHi or TLR agonists. Attenuated chemokine responses were associated with IRAK-1 protein degradation. Inhibition of RV-induced IRAK-1 degradation restored NTHi-stimulated IL-8 expression. Knockdown of TLR2, but not other MyD88-dependent TLRs, also restored IRAK-1, suggesting that TLR2 is required for RV-induced IRAK-1 degradation.
In conclusion, we demonstrate for the first time that RV infection delays bacterial clearance in vivo and suppresses NTHi-stimulated chemokine responses via degradation of IRAK-1. Based on these observations, we speculate that modulation of TLR-dependent innate immune responses by RV may predispose the host to secondary bacterial infection, particularly in patients with underlying chronic respiratory disorders.
Author Summary
Rhinovirus (RV) is responsible for the majority of common colds. RV infection is also associated with hospitalizations for lower respiratory tract illness, a significant proportion of which are accompanied by bacterial infections including acute otitis media, sinusitis and pneumonia. However, the mechanisms by which RV increases susceptibility to secondary bacterial infections are not understood. In this report, we demonstrate for the first time that RV infection promotes bacterial persistence of non-typeable Hemophilus influenzae (NTHi) in vivo, which was associated with reduced expression of neutrophil-attracting chemokines and neutrophil infiltration into the lungs. Further, RV infection attenuated NTHi or TLR2 or −5 agonist-stimulated chemokine responses in cultured bronchial epithelial cells and alveolar macrophages, suggesting that RV interferes with TLR-related innate immune responses. Next, we found that RV infection caused rapid degradation of IRAK-1, a key adaptor protein in the MyD88-dependent signaling. Inhibition of IRAK-1 degradation restored NTHi-stimulated chemokine responses in RV-infected bronchial epithelial cells. Finally, reductions in IRAK-1 were dependent on TLR2. Together, our results suggest that RV may increase the risk of acquiring secondary bacterial infection by attenuating TLR-dependent innate immune responses.
Citation: Unger BL, Faris AN, Ganesan S, Comstock AT, Hershenson MB, Sajjan US (2012) Rhinovirus Attenuates Non-typeable Hemophilus influenzae-stimulated IL-8 Responses via TLR2-dependent Degradation of IRAK-1. PLoS Pathog 8(10): e1002969. https://doi.org/10.1371/journal.ppat.1002969
Editor: Karen L. Mossman, McMaster University, Canada
Received: April 18, 2012; Accepted: August 28, 2012; Published: October 4, 2012
Copyright: © Unger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by National Institute of Health grants HL897720 and AT4793 to USS and HL81420 to MBH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Respiratory infection by one pathogen can alter the innate immunity to unrelated pathogens long after resolution of the first infection. This can affect the pathogen clearance and increase disease severity [1]. Severe illness or death due to bacterial infection following viral influenza is one of the most well-documented instances of this phenomenon [2]. Secondary bacterial infections (otitis media, sinusitis, pneumonia) have also been demonstrated following infection with respiratory syncytial virus, enterovirus and rhinovirus (RV) [3], [4], [5]. In addition, RV triggers exacerbations of chronic obstructive pulmonary disease and cystic fibrosis, conditions in which the airways are chronically colonized with bacteria [6], [7].
Most studies examining this problem to date have been focused on understanding the molecular mechanisms by which influenza virus predisposes host to secondary bacterial infection. Influenza is a lytic virus which causes extensive damage to the airway epithelium, thereby increasing exposure of bacterial receptors and inducing apoptotic cell death of macrophages and neutrophils [8], [9], [10]. IFN-γ production during influenza infection decreases the phagocytic and anti-bacterial capacity of alveolar macrophages [11]. Immunosuppressive cytokines such as IL-10 and TGF-β produced after influenza virus infection have been proposed to modify the initial chemokine response to subsequent bacterial infections [12], [13]. Glucocorticoids induced by influenza virus suppress pro-inflammatory cytokine responses to secondary bacterial challenge [14]. In addition to immunosuppressive molecules, antiviral proteins (such as IFNs) produced during viral infections attenuate initial KC/CXCL-1 and MIP2/CXCL2 responses to secondary pneumococcal challenge, resulting in increased persistence of bacteria and death in mouse models of infection [15]. Influenza virus also desensitizes TLR receptors in vivo and decreases pro-inflammatory cytokine responses to bacterial ligands long after the viral infection resolves [16].
Unlike influenza virus, RV does not cause excessive cell damage. Yet, RV infection has been shown to precede otitis media and acute lower respiratory tract infections requiring hospitalization, each of which are associated with bacterial infection [3], [4], [5]. A handful of studies have demonstrated that RV infection enhances bacterial adherence by increasing the expression of host molecules that serves as receptors for bacteria, such as ICAM-1, platelet-activating factor receptor and carcinoembryonic antigen-related cell adhesion molecule [17], [18]. RV infection was also shown to promote internalization of S. aureus into non-fully permissive lung epithelial cells [19]. In addition, RV infection disrupts barrier function and promotes transmigration of bacteria across the polarized airway epithelium [20], [21]. RV was recently shown to attenuate cytokine responses to subsequent challenges with two bacterial products, LPS and lipoteichoic acid, in alveolar macrophages [22]. However, the consequences of such RV-induced chemokine suppression on subsequent bacterial infection have not been demonstrated in vivo or in vitro. Further, the mechanism by which RV infection suppresses cytokine responses to subsequent TLR stimulation has not been determined.
In the present study, we show for the first time that RV promotes persistence of non-typeable Hemophilus influenzae (NTHi) by suppressing neutrophil-attracting chemokine responses. We also demonstrate that RV suppresses NTHi-induced IL-8 expression in airway epithelial cells and alveolar macrophages by inducing TLR2-dependent degradation of IRAK-1.
RV infection promotes NTHi persistence in vivo by suppressing chemokine expression and neutrophil recruitment
Major group rhinovirus, such as RV39, which binds to ICAM1 does not infect murine cells due to species specific variations in the ICAM-1 D1 extracellular Ig domain [23]. Previously, we have demonstrated the feasibility of infecting mice with RV1B, a minor group virus, which binds to low-density lipoprotein family receptors [24]. Therefore, in these experiments we used minor group virus, RV1B. Mice were infected with sham or RV1B by the intranasal route and two days later superinfected with NTHi by the intratracheal route. Chemokine expression and bacterial load in the lung were assessed 6 h and 1, 3 and 7 days post-NTHi infection. Although, there was no difference in the lung bacterial load between sham/NTHi and RV1B/NTHi groups at 6 and 24 h post-NTHi infection (Figure 1A), RV1B/NTHi group showed significantly less airway and interstitial neutrophils than sham/NTHi group at these time points (Figure 1B and 1C). While mice in sham/NTHi group cleared all bacteria by 72 h post-infection, RV/NTHi-infected animals showed bacteria in their lungs at low levels which were associated with increased number of airway and interstitial neutrophils. By 7 days post-NTHi infection, RV/NTHi-infected animals cleared all bacteria from their lungs and showed neutrophils counts similar to uninfected animals. Compared to sham-infected mice, RV-, sham/NTHi- and RV/NTHi-infected animals showed significant increases in both airway and interstitial lymphocyte counts 3 and 7 days post-NTHi infection (Supplemental Figure S1A and S1B). However, there was no difference between RV, sham/NTHi and RV/NTHi groups. Only the RV/NTHi group showed a significant increase in the number of macrophages/monocytes 3 and 7 days post-NTHi infection compared to sham-infected mice (Supplemental Figure S1C and S1D).
BALB/C mice were infected with RV1B or sham by intranasal route. Two days later, mice were infected with NTHi or treated with PBS via intratracheal route and sacrificed at 6 h, 1 day, 3 days or 7 days post-NTHi infection. (A) Mice were sacrificed and lungs collected aseptically, homogenized in sterile PBS and 10 fold serial dilutions of lung homogenates were plated to determine bacterial density. (B) Cytospins of BAL cells and (C) lung digests enriched for leukocytes were prepared, stained with Diffquick and number of neutrophils were counted. Data in A represent mean and range and in B and C, mean±SD (n = 4–9, * p≤0.05, two-way ANOVA, different from sham; # p≤0.05, two-way ANOVA, different from sham/NTHi infected animals).
Histologic evaluation of lung sections, revealed diffuse neutrophilic inflammation in both the sham/NTHi and RV/NTHi groups 24 h post-NTHi infection, but the latter group showed comparatively less inflammation (Figure 2A and 2C). While sham/NTHi-infected animals resolved inflammation completely by 3 days post-NTHi infection, mice in the RV/NTHi group showed a persistence of neutrophilic inflammation (Figure 2B and 2D). RV/NTHi-infected mice showed a resolution of lung inflammation 7 days post-NTHi infection, correlating with clearance of bacteria from the lungs (Figure 2E).
Mice were infected with sham or RV followed by NTHi as described in Figure 1. (A and B) Hematoxylin and eosin stained lung sections from Sham/NTHi-infected mice at 1 and 3 days post NTHi infection respectively. (C to E) Hematoxylin and eosin stained lung sections from RV1B/NTHi-infected mice at 1, 3 and 7 days post NTHi infection respectively. Inserts in the top left corners of A, C and D are magnified view of area marked in respective panels showing neutrophils. (Fand G) Chemokine levels in lung homogenate supernatants were determined by ELISA. Images in A to E are representative of 3 to 4 animals per group. Data in F and G represent mean±SD (n = 4–9, * p≤0.05, two-way ANOVA, different from sham; # p≤0.05, two-way f ANOVA, different from sham/NTHi infected animals).
Next we measured the levels of neutrophil-attracting chemokines in the lung homogenate supernatants. The RV/NTHi group showed a significant reduction in chemokine levels of KC/CXCL1 and MIP-2/CXCL2 compared to the sham/NTHi group (Figure 2F and 2G) at 6 and 24 h post-NTHi infection. However at 72 h post-NTHi infection, KC and MIP-2 levels were higher in the RV/NTHi group than the sham/NTHi group. Both KC and MIP-2 levels returned to normal levels by 7 days in both the sham/NTHi and RV/NTHi groups. These results imply that RV infection may suppress initial chemokine responses to subsequent bacterial challenge thereby reducing the neutrophil infiltration required for optimal bacterial clearance.
RV infection suppresses NTHi-stimulated IL-8 in bronchial epithelial cells
To understand the underlying mechanisms by which RV suppresses chemokine responses to subsequent NTHi infection, we performed in vitro studies using bronchial epithelial cells and confirmed key results in mouse alveolar macrophages. A human bronchial epithelial cell line (BEAS-2B cells) was infected with RV39 (a major group RV), RV1B or sham. Cells were then infected with NTHi and IL-8 protein in the cell culture supernatants was measured (Figure 3A and 3B). As observed previously, infection with RV or UV-RV stimulated IL-8 production in airway epithelial cells [25]. There was no difference between cells treated with RV and UV-RV. We have previously shown that RV binding/endocytosis is sufficient for stimulation of IL-8 expression in airway epithelial cells during the early phase of infection [25]. Cells infected with sham followed by NTHi showed significantly higher IL-8 levels compared to cells infected with sham or RV alone. In contrast, cells infected with RV39 or RV1B followed by NTHi showed IL-8 levels that were not significantly different from cells infected with RV alone. RV/NTHi-infected cells also showed significantly less IL-8 than sham/NTHi-infected cells, indicating that prior infection with RV suppresses NTHi-induced IL-8 production. Replication-deficient UV-irradiated RV had a similar effect. This was not a result of increased cell death, as there was no difference in LDH release or Annexin V/propidium iodide staining between sham/NTHi- and RV/NTHi-infected cells (data not shown).
(A and B) BEAS-2B cells grown to 90% confluence were infected with sham, or RV39, UV-RV39, RV1B, or UV-RV1B at MOI of 1 and incubated at 33°C for 90 min. Infection media was replaced with fresh media, and incubation continued for another 22 h. Cells were then infected with NTHi (10 MOI) or treated with media, incubated for 3 h at 37°C and IL-8 in the media was assayed for IL-8 protein by ELISA. (C and D) Mucociliary-differentiated airway epithelial cells were infected apically with sham, or RV39, UV-RV39, RV1B, or UV-RV1B at MOI and incubated for 24 h. Cells were then infected with NTHi at MOI of 10 and IL-8 in the basolateral medium was determined. (E and F) BEAS-2B cells infected with RV39 or sham were infected with FITC-conjugated NTHi at MOI of 10, incubated for 1 h at 37°C, washed to remove unbound bacteria and analyzed by flow cytometry to determine binding of bacteria to cells. E and F shows representative histograms and mean fluorescence intensity respectively. Data represents mean ± SEM from 2–3 experiments performed in duplicate or triplicates (* p≤0.05, ANOVA, different from sham; #p≤0.05, ANOVA, different from sham/NTHi).
To examine whether RV infection suppresses NTHi-induced IL-8 expression in well-differentiated primary airway epithelial cells, we infected these cells apically with RV39 or RV1B, incubated for 24 h and then infected with NTHi. Levels of IL-8 in the basolateral media were determined 6 h after NTHi infection. RV/NTHi-infected cells showed lower levels of IL-8 than the sham/NTHi-infected cells indicating that RV suppresses NTHi-stimulated IL-8 in primary cells (Figure 3C and 3D). Cells infected with UV-irradiated RV39 or RV1B also suppressed the IL-8 response to subsequent NTHi infection.
Pre-infection with RV, does not affect NTHi binding to bronchial epithelial cells
To investigate whether the suppressed IL-8 response to NTHi in RV-infected bronchial epithelial cells was due to decreased NTHi binding, we determined the binding of FITC-labeled NTHi to sham- or RV39-infected cells. Compared to uninfected cells, both sham/NTHi and RV39/NTHi infected cells showed a significant shift of histogram to the right and increased mean fluorescence intensity (MFI) indicating NTHi binding (Figure 3E and Figure 3F). Compared to sham/NTHi infected cells, RV39/NTHi infected cells showed further rightward shift of histogram and increased MFI, implying that prior infection with RV39 increases binding of bacteria to the cells. These results indicate that the suppressed IL-8 response in RV/NTHi-infected cells is not due to decreased bacterial binding.
RV infection reduces IL-8 responses to subsequent challenge with TLR agonist
Bacterial recognition by TLR2 and 5 contributes to IL-8 production in bronchial epithelial cells [26]. Therefore we examined whether RV suppresses TLR2- or TLR5-induced IL-8 responses in bronchial epithelial cells. BEAS-2B cells infected with sham and then challenged with a TLR2 (Pam3CSK4) or TLR5 (flagellin) agonist showed significant increases in IL-8 levels compared to sham alone infected cells (Figure 4A). Cells infected with RV39 followed by TLR2 or TLR5 challenge showed significantly lower IL-8 responses compared to similarly-challenged sham-infected cells.
(A) BEAS-2B cells were infected with RV39 or sham as described under figure 2 and then treated with Pam3CSK4 (TLR2 agonist), or flagellin (TLR5 agonist) and incubated for 6 h. IL-8 protein levels in the cell culture supernatants were determined by ELISA. (B, C and D) BEAS-2B cells were infected with RV39 or sham, blocked and incubated with antibodies to TLR2 or TLR5 followed second antibody conjugated with Alexafluor-488 and analyzed by flow cytometry. Histograms are representative of three independent experiments. Data in A and D represents mean ± SEM calculated from 3 independent experiments performed in duplicate (* p≤0.05, ANOVA, different from sham; #, p≤0.05, ANOVA, different from sham/TLR2 or sham/TLR5 treated cells).
TLR2 and TLR5 expression is not decreased by RV infection
RV-induced reductions in TLR-mediated IL-8 expression may relate to either reduced expression of TLRs or disruption of MyD88-dependent signaling, which is common for both TLR2 and 5. To assess the effect of RV infection on TLR expression, sham- or RV-infected cells were incubated with normal IgG or antibodies specific to TLR2 or TLR5 and analyzed by flow cytometry. BEAS-2B cells expressed both TLR2 and 5 on their surface, which were not significantly altered in RV39-infected cells (Figure 4B to 4D). These results suggest that suppression of TLR2 or TLR5 agonist-stimulated IL-8 in RV39-infected cells is not due to reduced TLR expression.
IRAK-M levels are enhanced in RV-infected cells
IRAK-M (also known as IRAK-3) is a well-described inhibitor of MyD88-dependent TLR signaling [27]. To investigate the possible role of IRAK-M in the suppression of NTHi-, or TLR2- or TLR5 agonist-induced IL-8, IRAK-M expression was assessed in RV39-infected BEAS-2B cells. RV significantly increased IRAK-M levels compared to sham controls (Figure 5A and 5B), suggesting that IRAK-M may be responsible for the observed suppression of NTHi- or TLR2 or 5 agonist-induced IL-8 expression in RV- infected cells. To examine this possibility, we transfected BEAS-2B cells with non-targeting or IRAK-M siRNA and then infected with either sham or RV. Western blot analysis showed that compared to NT–siRNA, IRAK-M siRNA transfection reduced IRAK-M expression by 83 and 77% in sham- and RV-infected cells respectively, an indication of efficient knockdown of IRAK-M in these cells (Figure 5C). However, knockdown of IRAK-M did not reverse the suppression of NTHi-stimulated IL-8 in RV-infected cells (Figure 5D). Similar results were observed when cells were challenged with TLR2 or TLR5 agonists instead of infecting with NTHi (data not shown). These data suggest that although RV increases IRAK-M expression, factors other than IRAK-M contribute to RV-induced suppression of NTHi-stimulated IL-8 response in bronchial epithelial cells.
(A and B) BEAS-2B cells were infected with sham, RV39 or UV-RV39 as described under Figure 2. Aliquots of total cell lysates corresponding to equal amount of protein from were subjected to Western blot analysis with IRAK-M antibody. (A) Representative blot showing IRAK-M expression. (B) Band intensities were quantified by NIH image and levels of IRAK-M was normalized to β-actin and expressed as fold increase over sham infected cells. (C) BEAS-2B cells were transfected with non-targeting (NT)- or IRAK-M siRNA and knockdown of IRAK-M was confirmed by Western blot analysis. (D) Similarly transfected cells were infected with sham or RV followed by NTHi as described in Figure 3 and IL-8 determined in the media. Data represents mean±SEM calculated from 3 independent experiments performed in triplicate (* p≤0.05, ANOVA, different from sham; #p≤0.05, ANOVA, different from sham/NTHi).
IRAK-1 expression is reduced in RV infection
It was recently shown that IL-1β and MyD88 are partially required for RV-induced IL-8 expression in BEAS-2B cells [28]. IRAK-1 undergoes proteosomal degradation following activation of the IL-1/TLR signaling pathway, thereby inducing a state of tolerance to subsequent TLR stimulation [29]. It is therefore conceivable that RV infection induces degradation of IRAK-1, leading to the attenuation of TLR-mediated IL-8 production, irrespective of IRAK-M status. To test this hypothesis, we determined the levels of IRAK-1 in BEAS-2B cells infected with either sham, RV39 or UV-irradiated RV39. Infection with RV39 and UV-RV39 each decreased IRAK-1 protein levels significantly 24 h after incubation (Figure 6A and 6B). RV39 infection decreased IRAK-1 levels as early as 4 h post-infection (Figure 6C and 6D). UV-RV39 also reduced IRAK-1 protein levels, indicating that the changes in IRAK-1 protein abundance do not require viral replication. Similar decreases in IRAK-1 were observed in BEAS-2B cells infected with RV1B (Figure 6E and 6F) and primary airway epithelial cells infected with RV39 or RV1B (Figure 6G and 6H).
(A and B) BEAS-2B cells were infected with sham, RV39, or UV-RV39, incubated for 24 h and the cells were lysed in RIPA buffer. The cell lysates containing equal amounts of protein was subjected to Western blot analysis with IRAK-1 antibody. (C and D) BEAS-2B cells were infected with sham or RV39 and cells were lysed at 4, 12 and 24 h post-infection and the lysates subjected to Western blot analysis with IRAK-1 antibody. (E and F) BEAS-2B cells infected with sham, UV-RV1B or RV1B were examined for IRAK-1 levels 24 h post- infection by Western blot analysis. (G and H) Mucociliary differentiated cells were infected apically with sham, RV39 or RV1B and IRAK-1 levels were determined 24 h later by Western blot analysis. Images are representative of 2–4 independent experiments. (B, D, F and H) Band intensities of IRAK-1 were normalized to β-actin and expressed as fold change over respective sham control. Data represents mean ± SEM calculated from 3 to 4 independent experiments (* p≤0.05, ANOVA, different from sham).
To determine whether IRAK-1 is required for NTHi-induced IL-8 expression, BEAS-2B cells transfected with non-targeting or IRAK-1 siRNA were infected with NTHi and the IL-8 response was determined 3 h later. Immunoblotting showed complete knockdown of IRAK-1 in cells transfected with IRAK-1 but not in non-targeting siRNA (Figure 7A). NTHi-induced IL-8 was significantly reduced in cells transfected with IRAK-1 siRNA compared to NT-siRNA-transfected cells (Figure 7B). These results imply that IRAK-1 is required for NTHi-induced IL-8 expression, consistent with the notion that reduced IRAK-1 protein levels contribute to RV-induced suppression of NTHi-induced IL-8 expression.
BEAS-2B cells were transfected with non-targeting (NT)- or IRAK-1 siRNA and grown for 3 days. (A) Cell lysates were prepared from transfected cells and subjected to Western blot analysis to confirm knockdown of IRAK-1. (B) Transfected cells were infected with NTHi, incubated for 3 h and IL-8 in the media determined. BEAS-2B cells were infected with sham or RV39 in the presence or absence of 5 µM lactacystin and incubated for 24 h. (C and D) Cells were either harvested for determination of IRAK-1 expression by Western blot analysis and band intensities of IRAK-1 were normalized to β-actin. (E) Cells were then infected with NTHi and IL-8 in the media was examined. Western blot images are representative of 3 experiments. Data in B, D and E represents mean±SEM calculated from 3 independent experiments performed in triplicate (* p≤0.05, ANOVA, different from sham; †, p≤0.05, ANOVA different from NT-siRNA transfected cells; # p≤0.05, ANOVA, different from DMSO treated RV/NTHi-infected cells).
To determine whether inhibition of IRAK-1 degradation in RV-infected cells restores NTHi-stimulated IL-8 expression, we infected cells with RV39 in the presence of proteosomal inhibitor lactacystin and examined IRAK-1 and IL-8 levels. In DMSO-treated cells (vehicle control), RV39 infection decreased IRAK-1 protein levels significantly (Figure 7C and 7D) and also suppressed NTHi-stimulated IL-8 (Figure 7E). By contrast, pre-treatment with lactacystin (5 µM) prevented RV-mediated reductions in IRAK-1 protein abundance and NTHi-induced IL-8 expression. These results show that IRAK-1 degradation caused by RV contributes to the reduced IL-8 response to subsequent NTHi infection.
IL-1 receptor antagonist (IL-1ra) does not reverse suppression of NTHi-stimulated IL-8 in RV infected cells
Since RV has been shown to activate MyD88-dependent signaling via IL-1β [28], we hypothesized that IL-1β is required for the suppression of NTHi-stimulated IL-8 in RV-infected cells. To test this hypothesis, BEAS-2B cells were infected with sham or RV in the presence or absence of IL-1ra. Cells were then infected with NTHi and IL-8 measured in the cell culture supernatant. IL-1ra did not reverse the suppression of NTHi-stimulated IL-8 caused by RV, even at high concentration (Supplemental Figure S2A). In addition, IL-1ra failed to restore the levels of IRAK-1 in RV-alone infected cells (Supplemental Figure S2B). Together, these results suggest that RV-induced IRAK-1 degradation is triggered independently of IL-1β.
RV-induced TLR2 activation is sufficient for reduction in IRAK-1 protein levels
We examined the contribution of the MyD88-dependent TLRs TLR2, −4, −5, −7 and −8 to RV-induced IL-8 expression. BEAS-2B cells were transfected with non-targeting siRNA or gene specific siRNA to TLR2, TLR4, TLR5, TLR7 or TLR8 and infected with RV39. IL-8 mRNA and protein levels were determined 24 h post-infection. TLR2 siRNA-transfected cells infected with UV-RV or RV showed significantly lower IL-8 mRNA and protein than similarly-infected non-targeting siRNA-transfected cells (Figures 8A and 8B). TLR8 siRNA-transfected cells showed a small (20%) but significant reduction in RV-induced IL-8 mRNA, but not protein levels. Knockdown of TLR4 TLR5, or TLR7 had no effect on RV or UV-RV-induced IL-8 responses either at the mRNA or at protein levels. Next we assessed the knockdown of each TLR by quantifying mRNA by qPCR (Figure 8C) and protein by flow cytometry (Supplemental Figure S3). BEAS-2B cells showed expression of TLR 2, −4, and −5 at both the mRNA and protein level, and transfection of gene-specific siRNA knocked down the expression. BEAS-2B cells did not express TLR7. Cells expressed TLR8 at very low levels and expression was completely inhibited in cells transfected with TLR8 siRNA.
BEAS-2B cells were transfected with NT, TLR2, TLR4, TLR5, TLR7 or TLR8 siRNA and grown for 2 days. (A and B) Cells were infected with sham, UV-RV39 or RV39 and incubated for 24 h. mRNA and protein levels of IL-8 was determined by qPCR and ELISA respectively. (C) Knockdown of TLRs was confirmed by qPCR. Data is normalized to GAPDH and is representative of three independent experiments. (D) Cells transfected with NT-, TLR2 or TLR8 siRNA were infected with sham or RV39 as above and cell lysates subjected to Western blot analysis with IRAK-1 antibody. (E) Band intensities of IRAK-1 were normalized to β-actin and expressed as fold increase over respective NT/sham control. Image in D is representative of 3 independent experiments. Data in A, B and E represents mean±SEM calculated from 3 independent experiments performed in triplicate (* p≤0.05, ANOVA, different from sham; # p≤0.05, ANOVA, different from NT-siRNA transfected cells).
Since only knockdown of TLR2 and TLR8 reduced RV-induced IL-8 responses, we examined the levels of IRAK-1 protein in TLR2 or TLR8 siRNA-transfected cells after RV infection. Compared to non-targeting siRNA-transfected cells infected with RV39, similarly-infected TLR2 siRNA-transfected cells showed IRAK-1 levels similar to sham-infected cells (Figure 8D and 8E). In contrast TLR8 siRNA transfected cells infected with RV showed IRAK-1 degradation similar to cells transfected with NT siRNA. These results imply that TLR2, but not TLR8 is required for the RV-induced reduction in IRAK-1 protein levels.
To assess whether TLR2 or TLR8 knockdown restores NTHi-stimulated IL-8 expression in RV-infected cells, cells transfected with non-targeting, TLR2 or TLR8 siRNA were infected with sham or RV39 and then infected with NTHi. Not surprisingly, TLR2 knockdown reduced NTHi-stimulated IL-8 (Supplemental Figure S4), as NTHi-induced IL-8 expression is TLR2-dependent [30]. On the other hand, TLR8 knockdown had no effect on NTHi-stimulated IL-8 and did not reverse the suppressive effect of RV on NTHi-stimulated IL-8.
Alveolar macrophages infected with RV1B show decreased IRAK-1 expression
RV infection has been shown to desensitize TLR4-dependent signaling in alveolar macrophages [22]. We therefore assessed the effects of RV1B infection on NTHi-stimulated cytokine expression and IRAK-1 protein levels in MH-S mouse alveolar macrophages. MH-S cells were infected with sham, UV-RV1B or RV1B and incubated for 24 h. Cells were then infected with NTHi and levels of KC, MIP-2 and TNF-α in the medium were determined 6 h later. Both UV-RV1B/NTHi and RV1B/NTHi-infected cells showed significantly less KC, MIP-2 and TNF-α than the sham/NTHi-infected cells (Figure 9A–9C). On a similar note, RV1B-infected cells also showed decreased KC and TNF-α production in response to secondary challenge with TLR2 (Figure 9D–9F) or TLR5 agonists (Figure 9G–9I). Compared to sham-, UV-RV1B and RV1B-infected cells also showed reduced IRAK-1 protein levels (Figure 10A and 10B), indicating that RV-induced IRAK-1 degradation may be responsible for reduced cytokine production in alveolar macrophages stimulated with bacteria or TLR agonists.
MH-S cells were infected with sham, RV1B or UV-RV1B. Cells were then challenged with (A to C) NTHi, (D to F) Pam3CSK4 or (G to I) flagellin and protein levels of KC, MIP-2 and TNF-α in the medium was measured after 3 (for TNF-α) or 6 h (for KC and MIP-2) post challenge by ELISA. Data represent mean±SEM calculated from 3 independent experiments performed in duplicates (* p≤0.05, ANOVA, different from sham; # p≤0.05, ANOVA, different from sham/NTHi infected cells).
(A and B) MH-S cells were infected with sham, RV1B or UV-RV1B as described in figure 2 and incubated for 24 h. Cells were lysed in RIPA buffer and aliquots of cell lysates containing equal amounts of protein was subjected to Western blot analysis with IRAK-1 antibody. (C and D) Mice were infected with sham or RV1B, sacrificed two days later and lung lysates subjected to Western blot analysis to determine IRAK-1 expression. (A and C) Image presented is a representative of three independent experiments and 3 different animals respectively. (B and D) Band intensities of IRAK-1 were normalized to β-actin and expressed as fold increase over sham control. Data represents mean and SEM calculated from 3 independent experiments in B and 4 animals in D (* p≤0.05, ANOVA, different from sham).
RV1B infection reduces IRAK-1 protein levels in vivo.
Next, we assessed whether RV infection affects IRAK-1 protein levels in vivo. Mice were infected with sham or RV1B and 48 h later, IRAK-1 protein was determined in the whole lung homogenates by Western blot analysis. Compared to sham infected mice, RV1B infected mice showed a significant reduction in IRAK-1 levels (Figure 10C and 10D). Together, these results indicate that RV-induced reductions in IRAK-1 may attenuate chemokine production in response to secondary bacterial infection via desensitization of MyD88-dependent TLR signaling.
In this study, we demonstrate for the first time that RV infection delays bacterial clearance in vivo. Delayed bacterial clearance was accompanied by attenuated expression of the neutrophil-attracting chemokines KC/CXCL1 and MIP-2/CXCL2. We also show that, despite increasing bacterial binding, RV suppresses chemokine responses to subsequent NTHi infection in cultured bronchial epithelial cells. RV infection also attenuated TLR2- and TLR5-stimulated chemokine responses in cultured bronchial epithelial cells and alveolar macrophages, suggesting that RV infection interferes with TLR-dependent innate immune defenses. Most importantly, we define a mechanism by which RV infection desensitizes TLR signaling in both bronchial epithelial cells and alveolar macrophages. We demonstrate that RV infection causes degradation of IRAK-1, a key adaptor protein in MyD88-dependent TLR signaling, thereby suppressing chemokine responses to subsequent NTHi challenge. Further, we show that IRAK-1 degradation was dependent on TLR2, but not on other TLRs which activate MyD88-dependent signaling. Based on these observations, we speculate that suppression of TLR-dependent innate immune responses increases the risk of acquiring secondary bacterial infection and promotes invasiveness of bacterial flora that is present in the nasopharynx/lower airways of CF and COPD patients.
Although there is clinical evidence indicating that RV infection precedes bacterial infection-associated disease exacerbations [3], [4], [5], until now there were no experimental data demonstrating an increased risk of bacterial infection following RV infection in vivo. Using a novel mouse model of RV infection that we developed [24], [31], we found that RV infection promotes NTHi persistence in the lungs, likely by attenuating KC and MIP-2 expression and impairing recruitment of neutrophils to the airways. Appropriate neutrophil recruitment is required for bacterial clearance, particularly under circumstances that exceed the antimicrobial capacity of airway epithelial cells and alveolar macrophages. Mice with a delayed neutrophil response fail to clear P. aeruginosa efficiently from their lungs [32]. Recently, influenza virus was also shown to suppress KC and MIP-2 responses to subsequent bacterial infection; suppression was dependent on virus-stimulated type I interferons [15]. Since RV infection also stimulates type I interferon responses both in vivo and in airway epithelial cells in vitro [24], [33], [34], [35], [36], it is plausible that RV suppresses chemokine responses via this mechanism. However, this is unlikely, because replication-deficient UV-irradiated RV, which does not stimulate expression of type I interferons, also attenuated chemokine response to NTHi infection in vitro.
Both bronchial epithelial cells and alveolar macrophages express TLRs. TLRs play a crucial role in the establishment of appropriate innate immune responses, including expression of chemokines, upon recognition of pathogen associated molecular patterns. Previously, RV infection was shown to suppress subsequent cytokine responses to LPS, a TLR4 ligand in human alveolar macrophages [22]. Suppression was dependent on viral replication. In contrast, we found that attenuation of TLR-dependent signaling in both bronchial epithelial cells and alveolar macrophages was independent of viral replication. These data suggest that suppression is initiated by an early event in the viral life cycle, for example, binding or endocytosis. This is not surprising, because RV binding itself has been shown to activate signaling mechanisms leading to phosphatidylinositol 3-kinase activation, IL-8 expression and generation of reactive oxygen species [25], [37].
In the present study, we found that RV infection not only attenuated chemokine expression due to subsequent challenge with NTHi infection, but also to TLR2 and TLR5 ligands. NTHi stimulates TLR2 signaling leading to pro-inflammatory cytokine expression [30]. In addition, both TLR2 and TLR5 initiate a MyD88-dependent signaling pathway leading to chemokine and pro-inflammatory cytokine expression. We therefore hypothesized that RV attenuates MyD88-dependent TLR signaling. Accordingly, we found that RV infection significantly increased IRAK-M while dramatically reducing the levels of IRAK-1 protein in both airway epithelial cells and murine alveolar macrophages. IRAK-M is thought to be a negative regulator of MyD88-dependent TLR signaling [38]. However, genetic silencing of IRAK-M did not restore NTHi-induced chemokine expression in RV-infected airway epithelial cells, suggesting that IRAK-M does not contribute to RV-induced attenuation of IL-8 expression. This is not entirely unexpected, as IRAK-M has been demonstrated to play a vital role in the suppression of a non-canonical TLR signaling pathway that relies on activation of NF-κB-inducing kinase rather than IκB kinase [39].
In contrast, blocking of RV-induced reduction in IRAK-1 by using a proteasomal inhibitor restored NTHi-induced IL-8 responses in RV-infected bronchial epithelial cells, indicating a role for IRAK-1. Previously, it has been shown that post-translational modification of IRAK-1, such as hyperphosphorylation and ubiquitination that occurs following MyD88-dependent TLR or IL-1 signaling, leads to IRAK-1 degradation [29]. IRAK-1 degradation constitutes a mechanism for limiting exaggerated innate immune responses. In the present study, we found that, in vitro, IRAK-1 is degraded as early as 4 h post-RV infection and the IRAK-1 protein levels remain low for at least 24 h. Pre-treatment with lactacystin prevented reductions in IRAK-1 protein abundance, suggesting that RV promotes proteasomal degradation of IRAK-1. Reduced IRAK-1 was also observed in mice infected with RV 48 h post-infection. These observations suggest that RV induces IRAK-1 degradation via the activation of MyD88-dependent IL-1/TLR signaling cascades. However, since lactacystin can also inhibit proteasomal degradation of other proteins of TLR pathway, further studies using IRAK-1 mutants which are not amenable to ubiquitation and proteasomal degradation are necessary to further confirm the specific role of IRAK-1 in RV-induced suppression of NTHi-stimulated chemokine responses.
Recently, RV infection of BEAS-2B cells was shown to elicit low levels of IL-1β release, which in turn induced MyD88-dependent potentiation of IL-8 production [28]. However, in the present study, inhibition of IL-1 receptor by IL-1ra neither blocked RV-induced degradation of IRAK-1 nor reversed suppression of NTHi-stimulated IL-8, suggesting that the IRAK-1 degradation that we observed is not due to RV-induced IL-1β signaling.
RV single-stranded RNA may activate MyD88-dependent TLR signaling directly via TLR7 and TLR8. In our studies we found that airway epithelial cells do not express TLR7 and express TLR8 at very low levels. Further, knockdown of TLR8 neither prevented RV-induced degradation of IRAK-1 nor reversed suppressive effects of RV on NTHi-stimulated IL-8. Similar to our results, recently it TLR7/8 agonist, imiquimod was found to be inefficient in activating airway epithelial cells suggesting that these cells may not express TLR7 or 8 [40]. On the other hand, it has been demonstrated that both intact and UV-irradiated RV-6 stimulate NF-κB transactivation in TLR2-transfected HEK-293 cells [41]. Since we observed that both replication-competent and replication-deficient RV were equally capable of causing IRAK-1 degradation, we posit that TLR2 plays a role in RV-induced IRAK-1 degradation. Consistent with this notion, we found that knockdown of TLR2 significantly reduced RV-induced IRAK-1. However, we could not determine the effect of TLR2 knockdown on suppression of NTHi-stimulated IL-8 in RV-infected cells, because NTHi also requires TLR2 to stimulate IL-8 [30]. Also, the precise mechanism by which RV activates TLR2 remains to be elucidated.
In summary, we have shown that RV suppresses NTHi-induced IL-8 expression in airway epithelial cells and alveolar macrophages by inducing TLR2-dependent degradation of IRAK-1. Our results suggest that RV may increase the risk of acquiring secondary bacterial infection by attenuating TLR-dependent innate immune responses.
Materials and Methods
Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, United States. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Michigan Medical School. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Tracheal and bronchial trimmings from donor lungs obtained at the time of double lung transplantation (which are otherwise discarded) were used for isolation of airway epithelial cells and the protocol was approved by the University of Michigan Investigational Review Board. Since the tissues were not collected for the purpose of isolation of cells and there was no documentation linking the tissue to the donor, the University of Michigan Investigational Review Board provided waiver for obtaining consent.
RV39 and RV1B was obtained from ATCC and used to generate viral stocks via infection of HeLa cells as previously described [25]. Virus titer was determined by assessing 50% tissue culture infectivity dose (TCID50)/ml. For production of UV-irradiated (UV-RV) RV, samples were irradiated using a CL-1000 crosslinker (UVP, Upland, CA) at 10 mJ/cm2 for 10 min on ice as described previously [33]. RV inactivated by this method is fully capable of binding to epithelial cells and undergoing endocytosis and stimulates IL-8 protein levels similar to intact RV. At the same time it does not stimulate replication-dependent interferons and interferon-dependent genes.
Bacterial culture and conditions
Nontypeable Haemophilus influenzae (6P5H) was isolated from a COPD patient during exacerbation (Kindly provided by T. Murphy, University of Buffalo, Buffalo, NY). Bacteria was maintained as a glycerol stock at −80°C and cultured as described previously [42].
Animals and infection
Normal 8 to 10 week-old BALB/C mice were infected with 50 µl of RV1B (1×108 TCID50T/ml) by intranasal route as described previously [24], [31]. Mice were then infected with 40 µl NTHi (1×109 colony forming units/ml) by intratracheal route [43] and sacrificed 6 h, 1 day or 3 days or 7 days post-NTHi infection. Lungs were collected under aseptic conditions, then homogenized in 2 ml sterile PBS. An aliquot of lung homogenate was 10 fold serially diluted and plated on chocolate agar plates to determine bacterial density. Lung homogenates were centrifuged and supernatants used for ELISA. Number of airway and tissue inflammatory cells was determined by counting cells in bronchoalveolar (BAL) fluid and lung digests respectively as described previously [34]. Briefly, after appropriate treatment, BAL was performed by instilling 1 ml PBS containing 5 mM EDTA 10 times. Cytospins prepared from BAL cells were stained with Diff-Quick and differential counts were determined by counting a minimum of 200 cells. To quantify the number of inflammatory cells in the tissues, lungs were digested with type IV collagenase for 1 h, lung digests were strained through 70-µm nylon mesh (BD Biosciences, San Jose, CA) and centrifuged. The cell pellet was treated with RBC lysis buffer (BD Biosciences) and subjected to density gradient centrifugation on 40% Percoll (Sigma-Aldrich, St. Louis, MO) to enrich for leukocytes. The total cell count was determined on a hemocytometer. Cytospins of these tissue leukocytes were stained with Diff-Quick and examined for differential cell counts. In some experiments, after appropriate treatment, lungs were inflation fixed and embedded in paraffin. Five micron thick sections were deparaffinized and stained with hematoxylin and eosin to evaluate lung inflammation.
Cell culture and infection
Human bronchial epithelial cell line (BEAS-2B; American Type Culture Collection, Manassas, VA) were grown in collagen coated plates using bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD)) as described previously [33]. Primary airway epithelial cells from normal subjects were cultured at air/liquid interface to promote differentiation into mucociliary phenotype [33], [42], [44]. Mouse alveolar macrophages (MH-S) (ATCC) cells were grown in RPMI media amended with L-glutamine and 10% serum.
BEAS-2B or MH-S cells grown to 90% confluence were infected with RV or UV-RV at multiplicity of infection (MOI) of 1 or equal volume of sham (media from uninfected HeLa cells) and incubated for 90 min at 33°C. Infection media was replaced with fresh media and the incubation continued for another 22 h. Cells were then infected with NTHi at MOI of 10 or treated with media alone, centrifuged at 500×g for 5 min and incubated at 37°C for 3 h. Media was collected for determination of IL-8 (for BEAS-2B cells) or KC, MIP-2 and TNF-α (for MH-S cells). In some experiments, cells were infected with RV in the presence of 5 µM lactacystin (Cayman chemicals, Ann Arbor, MI) or 10 to 100 ng/ml IL-1 receptor antagonist (PeproTech, Rocky Hills, NJ). Primary cells were infected apically with RV, UV-RV or sham at 1 MOI [45], incubated for 48 h and then cells were superinfected apically with NTHi at 10 MOI and basolateral media collected for IL-8 and cells for Western blot analysis.
TLR agonists
BEAS-2B cells were infected with RV, UV-RV or sham as described above. Cells were then challenged with TLR2 agonist, Pam3CSK4 (InvivoGen, San Diego, CA), or TLR5 agonist flagellin (ENZO Life Sciences, Inc., Farmingdale, NY), incubated for 6 h at 37°C and IL-8 determined in the cell culture supernatant.
SDS-PAGE and Western blotting
After relevant treatment, cells were washed with ice cold PBS and total cell lysates were prepared as described previously [46]. Briefly, cells were lysed in cold RIPA buffer containing complete protease inhibitors (Roche Diagnostics, Indianapolis, IN), lysates centrifuged and total protein determined in supernatants. Lungs harvested from mice were homogenized in 1 ml PBS conataining complete protease inhibitors and mixed with 2× RIPA buffer, sonicated for 3×10 seconds each, centrifuged and total protein determined in supernatants. Aliquots equivalent to equal amounts of protein were subjected to SDS-PAGE and protein transferred to nitrocellulose membrane. Membranes were blocked and probed with antibodies to IRAK-M, IRAK-1 (both from Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma-Aldrich, St. Louis, MO). Bound antibody was detected by using appropriate second antibody conjugated with horseradish peroxidase and chemiluminescent substrate. Band densities were normalized to β-actin or relevant total protein using NIH Image-J (NIH, Bethesda, MD).
Bacterial binding assay
Binding of bacteria to cells was determined using FITC-labeled NTHi as described previously [47]. Briefly, FITC-labeled NTHi was incubated with BEAS-2B cells infected with Sham, UV-RV or RV and incubated for 60 min. Cells were then washed and analyzed by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ).
Detection of TLR expression by flow cytometry
After relevant treatment BEAS-2B cells were incubated with blocking buffer followed by monoclonal antibodies to either TLR2, TLR4, TLR5, TLR7 or TLR8 (all from eBioscience, Inc., San Diego, CA) or normal mouse IgG (matched isotype control) conjugated with fluorescein isothiocyanate (FITC). For detection of TLR7 and TLR8 cells were permeabilized with 1% saponin for 30 min on ice prior to incubating with antibodies to TLR7 or TLR8. Cells were then washed and analyzed by flow cytometry [42].
Conditioned basolateral medium from cell cultures or lung homogenate supernatant was used to determine the protein levels of chemokines by ELISA (R&D systems, Minneapolis, MN) as described previously [45], [48].
Transfection of BEAS-2B cells
BEAS-2B cells were reverse transfected with 10 picomoles of non-targeting (NT), or ON-TARGETplus SMART pool siRNA specific to TLR2, TLR4, TLR5, TLR7, TLR8, IRAK-1 or IRAK-M (Dharmacon, Inc., Chicago, IL) and incubated for 48 h. Cells were then infected with sham, RV or NTHi as appropriate (described in results section). Knockdown of gene expression was confirmed by qPCR or Western blot analysis.
Results are expressed as means ± SEM. Data were analyzed by SigmaStat statistical software (Systat Software, Inc., San Jose, CA). One-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc analysis or two-way ANOVA was performed to compare more than two groups. To compare two groups, an unpaired t test with Welch's correction was used. If the data were not distributed normally data were expressed as range with median and analyzed by Mann-Whitney test. A value of p≥0.05 was considered significant.
Supporting Information
Figure S1.
RV/NTHi infection does not alter macrophage/monocyte and lymphocytes in mice. BALB/C mice were infected with RV1B or sham by intranasal route. Two days later, mice were infected with NTHi or treated with PBS via intratracheal route and sacrificed at 6 h, 1 day, 3 days or 7 days post-NTHi infection. Cytospins of BAL cells and leukocyte enriched fraction from lung homogenates were prepared, stained with Diffquick and number of (A and B) lymphocytes and (C and D) macrophage/monocytes were counted. Data represent mean±SD (n = 5, *p≤0.05, two-way ANOVA, different from sham-infected animals).
Figure S2.
RV induced reduction in IRAK-1 protein is not IL-1β-dependent. BEAS-2B cells were infected with sham or RV39 and incubated at 33°C for 90 min. Infection media was replaced with media or media containing IL-1ra and incubation continued for another 22 h. (A) Cells were washed once with fresh media and then infected with NTHi and IL-8 in the media was assessed after 3 h. Data represents mean±SEM calculated from 3 independent experiments performed in triplicates (* p≤0.05, ANOVA, different from sham; #p≤0.05, ANOVA, different from sham/NTHi). (B) Cells were lysed following RV infection and the lysates corresponding to equal amounts of protein was subjected to Western blot analysis with IRAK-1 antibody. Image is a representative example of 2 independent experiments.
Figure S3.
Gene specific siRNA to TLRs inhibits protein expression of respective TLRs. BEAS-2B cells were reverse transfected with NT-, TLR2, TLR4, TLR5, TLR7 or TLR8. After 48 h incubation, cells were either permeabilized (for TLR7 and TLR8) or not (TLR2, TLR4 and TLR5), incubated with antibodies to (A) TLR2, (B) TLR4, (C) TLR5, (D) TLR7 or (E) TLR8 or normal IgG conjugated with FITC and analyzed by flow cytometry. Image is a representative example of 2 independent experiments.
Figure S4.
Knockdown of TLR2 decreases NTHi-stimulated IL-8. BEAS-2B cells reverse transfected with NT- or TLR2 siRNA were grown for 2 days. Cells were infected with NTHi or sham infected with media, incubated for 3 h at 37°C and IL-8 the medium was determined. Data represents mean±SEM calculated from 3 independent experiments performed in triplicates (* p≤0.05, ANOVA, different from media control; #p≤0.05, ANOVA, different from cells transfected with NT siRNA and then infected with NTHi).
Author Contributions
Conceived and designed the experiments: BLU ANF USS. Performed the experiments: BLU ANF SG ATC. Analyzed the data: BLU ANF SG USS. Contributed reagents/materials/analysis tools: USS. Wrote the paper: BLU MBH USS.
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Join us as we visit sanctuaries and homes across the country to discover the animals most of us never have a chance to meet – America's farmed animals.
This collection of stories and photographs by rescuers, caregivers and animal lovers will show you a side of these animals you may not have thought possible.Far from the unthinking, unfeeling beings they are often portrayed as, the animals in Ninety-Five* reveal themselves to be as distinctive as the dogs and cats with whom many of us share our homes: complex creatures with remarkable personalities, depth, emotions, quirks, relationships and individuality.
From Justice, the steer who broke out of a truck on the way to the slaughterhouse and who is now the self-appointed "greeter" at his sanctuary, to Gilly, a small white hen rescued from a factory egg farm who found quiet happiness in a loving home, each animal has a compelling story that will captivate. Beautiful photos complement, allowing us the chance to get up close and personal, and to look into their eyes – the "windows of the soul" – and see the extraordinary spark of life they hold.
Some of the animals will make you chuckle, others will move you, some may bring tears to your eyes, and some will just give you something to think about.All of them are fascinating. Come with us on a journey to meet them…

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Daily Roundup: George Strait, Scotty McCreery + More
Friday was a really big day for George Strait. The King of Country turned 60 on May 18, and we celebrated right along with him with a list of little-known facts about the singer. Find those in today's Daily Roundup -- plus, find out when to tune in to watch Scotty McCreery return 'home' next week.
Each day, we bring you all of the latest country music news mixed with a little news from elsewhere around the web. Today, discover whether or not Jennifer Lopez is leaving 'American Idol' and learn which body part a Michigan teen discovered in his Arby's sandwich... if you dare.
Today on Taste of Country:
10 Things You Didn't Know About George Strait
Scotty McCreery to Return to 'American Idol' for Season Finale Performance
Taylor Swift Shares Creative 'Eyes Open' Lyric Video
Favorite George Strait Song – Readers Poll
'The Voice' May Get New Coaches Next Season
2012 CMT Music Awards Performers Include Carrie Underwood, Brad Paisley + More
Pudding Shots Recipe by Thom Shepherd
'Nashville' Picked Up by ABC, Show Trailer Hits the Web
Picture of the Week: Dierks Bentley and Eli Young Band Grab a Shotgun
Win a Pair of Justin Boots Autographed by Brandon Rhyder
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Notre Dame-West Haven remains perfect with shutout win over Darien
WEST HAVEN -- Notre Dame-West Haven made it a perfect seven-for-seven to start the season by defending its home ice with a 3-0 victory over previously unbeaten Darien.
After nearly two scoreless periods, Notre Dame-West Haven's William Vizzo finally tilted the game in his team's favor by scoring with just 29 seconds remaining in the second period for the 1-0 lead heading into the third.
"Obviously, it was huge. We needed a goal, and I just put the puck on net and it went in," Vizzo said. "It's a huge win. We want to have confidence going into Saturday against Prep."
After being outshot 15-8 in the first period, Darien (3-1-1) had the opportunity it was looking for early in the second, as Notre Dame-West Haven (7-0-0) took two minor penalties just 35 seconds apart, setting the stage for 1:25 of 5-on-3. Darien took three shots, including one each by Robbie Juterbock and Thomas Watters, but Notre Dame successfully killed the two-man advantage.
"We didn't expect to be this way because we lost six kids from last year, but the younger kids are stepping up," Notre Dame-West Haven coach Bill Gerosa said. "We have a lot of young kids on the team and we've played the FCIAC early. They've done a good job against us, and it helps us in the long run."
Darien goalie Michael Collins made several difficult saves, but his highlight of the night came with the game still scoreless and Notre Dame on the powerplay.
Brendan Morley controlled the puck at the Darien blue line and sent a pass to Vizzo, who was lurking next to the Darien net. Collins moved to his right for a potential shot, but Vizzo instead sent it back across the goal crease to Kyle Thibault, who was robbed when Collins dove back to his left to make a glove save.
Notre Dame-goalie Luciano Amatruda stopped all 20 of Darien's shots to earn the shutout, and he was also the beneficiary of a favorable bounce at the beginning of the third period. Trailing 1-0, Darien won a faceoff in the offensive zone before ringing a shot off the post.
"Maybe a bounce here and there and the tide turns our way. It was either Bergin or Knowlton off of a designed play. If that goes in, it's a 1-1 game," Darien coach Chris Gerwig said. "Our guys stayed focused and we battled back. It's a great measuring stick for us, and especially after we hadn't played a game in 10 days. Nobody likes to lose, but you always have to take some positive things out of losses."
Instead, the Green Knights maintained the lead, and seven minutes later, they increased it on a goal by Thibault.
Trailing 2-0, Darien pulled Collins with 1:40 remaining, but Notre Dame's Gregory Zullo notched the empty-netter with 1:15 on the clock to ice the game. Louis Iannotti assisted on all three goals.
"They gave us all we could handle," Gerosa said. "Between our goaltending and defense, we got the job done."
email@example.com; Twitter: @jchik17
Records: NDWH 7-0-0; D 3-1-1
Goals: ND--William Vizzo, Kyle Thibault, Gregory Zullo. Assists: ND--Louis Iannotti (3). Goalies: ND--Luciano Amatruda (20 saves); D--Michael Colon (28). Shots: ND--31; D--20.


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Tour de France tech: New aero kit at team time trial
Revamped BMC Timemachine TM01, Mavic wheels
BMC show off stealthy new Timemachine TM01
BMC took to the start line of the team time trial aboard a new Timemachine TM01 flagship aero bike. This revamped machine still wears a cutting-edge aesthetic but now has a far greater level of component integration, tweaked tube shapes, and easier fit adjustability than before.
As is the case with many of the latest dedicated time trial bikes, the TM01 is distinguished by its clean appearance. Aside from a few bits of wiring harness and very short sections of brake housing, there are no visible control lines anywhere on the bike. Everything is fed from the bars straight into the frame beneath a tidy cap behind the stem and the Shimano Dura-Ace Di2 battery is tucked safely away in a pocket inside the seat tube.
As on the original TT01, BMC retain the use of an external steerer tube but now the fork crown also houses an integrated brake caliper. Out back, a special set of mini V-brakes is neatly blended into the chainstays and effectively 'hidden' from the wind by a slight down tube extension. BMC also manage to carry over their trademark T-shaped top tube profile, but the rest of the frame is distinctly more angular, with a far less organic form than before – particularly where the dropped seatstays join with the seat tube.
BMC tuck a set of mini V-brakes underneath the chainstays on their new Timemachine TM01
Running along the sides of the fork blades, external fork steerer, seat tube, telescoping seatpost and seatstays is a subtle rib just behind the leading edge. According to BMC, this 'tripwire' feature creates just a hint of turbulent air that then helps smooth airflow over the rest of the tube. Trailing edges, on the other hand, are slightly truncated.
BMC are also emphasizing the new bike's easier adjustability. Aside from a pair of custom-molded fully one-piece front ends built only for the team, bar position can be adjusted via several bolt-on stem stubs, which can also be flipped over for additional positions or spaced out and angled via interchangeable shims and wedges.
To better accommodate the fit-divergent time trial and triathlon markets, the telescoping seatpost also offers four different offset positions. Other features include a BB30-compatible bottom bracket shell, built-in rotational stops on the top tube to prevent fork damage in a crash, and adjustable-length rear-entry dropouts. BMC chief technical officer Rolf Singenberger says production versions will be available beginning this September.
The modular stem on the redesigned BMC Timemachine TM01 allows for a wide range of bar positions with a single molded carbon fork structure
Mavic "CX01 Technology" minds the gap
Mavic-sponsored teams Garmin-Cervélo and Liquigas rocketed around Cholet with new 80mm-deep carbon tubular front wheels featuring a notably wide profile, carbon bodied hubs, bladed stainless steel spokes and – thank you! – externally accessible alloy nipples. Mavic were mum on the official name but taking a closer look at the blacked-out labels reveals that it'll be called the CXR 80 at its expected debut later this year at Eurobike.
Almost more intriguing than the wheel itself, however, was the 'CX-01' snap-on fairing that fills in the gap between the tire and rim bed. Mavic haven't offered up technical information on exactly how much of an aerodynamic advantage this may provide but we suspect it's rather modest. Regardless, it can't hurt, it seems relatively easy to install and also looks to be compatible with other tire makes and models.
Garmin-Cervélo and Liquigas-Cannondale team bikes were fitted with new 80mm-deep Mavic carbon tubular front wheels along with clever snap-on fairings
Lampre put new Wilier TwinFoil to the test at Le Tour
After a short stint on fairly generic aero bikes at last year's Tour de France – mechanics told us the old Cobb-designed frame ran into trouble with the UCI – Lampre gave their new UCI-approved Wilier TwinFoil aero machines a second major test at this year's Tour de France team time trial. One of the major distinguishing features of the new bike is the TT-specific TRP integrated mini-V rear brake.
This has been moved down below the chainstays behind the bottom bracket and its shape blends in nicely with the rest of the frame for smoother airflow. Relocating the brake also allowed for more dramatic seatstay shaping and like other companies that have made similar moves, Wilier push those stays further apart to supposedly minimize air turbulence created by the rear wheel.
The clever front derailleur tab on Lampre's new Wilier TwinFoil is adjustable for angle to better accommodate different chainring sizes and shapes
Shaping up front is fairly conventional, with deep fork blades and internally routed cables that enter on the sides of the aero-profile down tube. However, Wilier raise the top tube to better blend with the stem – a move Specialized made a while back on their old Transition. Other features include a telescoping aero carbon post with both an external clamp and internal wedge to secure it in place, sliding rear dropouts to tighten up the gap between the tire and seat tube, and a clever rotatable front derailleur mount to better accommodate bigger or non-round chainrings.
Stunningly sleek front ends with integrated Di2 shift buttons for Sky's Pinarello Graal
Team Sky's Pinarello Graals arrived in Cholet with new carbon fiber and aluminum cockpit setups that took excellent advantage of the engineering flexibility afforded by Shimano's Dura-Ace Di2 system. The fresh one-piece base carbon fiber bar and stem featured not only knifelike brake levers molded directly into the outer pods but also prototype Di2 shifter pods blended seamlessly into the rest of the structure with small, uncharacteristically rounded buttons – standard Shimano shifter pods were fitted to the extension ends.
Shimano Dura-Ace Di2 shift buttons are perfectly integrated into the base bars on Sky's Pinarello Graals. For the record, Shimano deny any involvement
There's no word on whether these particular bits will be made available to the public, and Shimano representatives who happened to be on site denied any involvement in the project. Assuming that's truly the case, this may very well be a Sky team exclusive. Additional features with the new Sky Graal front ends include sculpted, stackable aluminum spacers for extension and pad height, telescoping extension mounts with collet-type clamps, at least six extension width positions, internal cable routing and adjustable stem length.
FDJ debut fresh Lapierre aero machines
FDJ's new Lapierre Aerostorm bikes made their debut Tour de France appearance during the team time trial after first making the rounds at last year's trade shows. Certainly elements bear a resemblance to other machines: the tightly tucked and kinked chainstays (Scott Plasma), the rear brake caliper placement atop the chainstays and behind the bottom bracket shell (original Felt DA), and the raised top tube that presumably helps smooth out airflow off the back of the stem (Specialized Transition).
The FDJ team used Lapierre's new Aerostream during Stage 2
Other design features are most definitely unique to Lapierre, however, including the peculiar laid-back seat tube with its replaceable, bolt-on – and bolt upright – integrated seatmast with a two-position head. In addition, the fork is built with front loading horizontal tips for user-tunable rake and handling, and Shimano Di2 users will find a handy compartment in the down tube for the battery.
Specialized show off new TT3 aero helmet
Specialized-sponsored riders raced around Cholet using the company's latest TT3 aero helmet. As compared to the current TT2, the new model features a more rounded profile with fewer kinks to disrupt airflow, less angular earflaps, smaller rear vents, a lower rear cover, a bolt-on visor, and pluggable front vents for when reducing drag is of the utmost importance.
Specialized's new TT3 aero helmet features a revised shape with pluggable front vents and an optional bolt-on visor


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How many law schools are in Chicago? I only know of 5: Chicago, Northwestern, Loyola, UIC and John Marshall.. Thought about school in Chicago but had the mid west for undergrad and wasn't looking forward to the freeze.
Oh petitschoque I didnt peep that thread. Mind linking me?
Quote from: Andromeda on February 28, 2006, 04:14:19 PMI see there are also NIgerians on here. Anyone, Nigerians that is, planing on going to school in Chicago??You're Naija?I might be going to school in Chicago
I see there are also NIgerians on here. Anyone, Nigerians that is, planing on going to school in Chicago??
Pardon me if I'm being too inquisitive. Another question I have is do you guys ever have any issues with African Americans? I know people quickly assume you are African American. Your culture is different. Do you try to separate yourselves from them? Did you have any problems growing up? Any conflict with other Af-Am?
Not sure yet if I'll end up in Chicago, but I'm still thinking about it. You going there for sure?
Quote from: Andromeda on March 01, 2006, 07:23:46 AMYep! Wow! what tribe and city/town?QuoteNot sure yet if I'll end up in Chicago, but I'm still thinking about it. You going there for sure?I'm the same as you--not sure. I'm leaning toward UChicago because of it's reputation but then again I've heard so many reports about how ugly the campus is and the weather really is cold...I've been yoyo-ing back and forth between like 5 schools and I'm waiting to hear from one about money...
Yeah, I am worried about the weather the most. I've been out there to visit friends/family and the weather is just cooollld! I'm from Delta, and I grew up partly in Lagos, you?.
u'd have to be able to stomach below zero winters and 10 inch snow days without classes being cancelled in spite of it...the greatness of University of Chicago outweighs a few months of horrible weather...


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Once again, we want to give a big thanks to all of our volunteer recipe testers for your thoughtful comments and for helping us continue to strengthen our recipe recommendations. You can read each tester's comments by clicking through to the Community Picks recipes — the comments are at the top. And even if the recipe you tested wasn't chosen as a CP, please feel free to leave your testing notes in the recipe's comments section. Constructive criticism is always encouraged!
Garlic Scape Beef Satay with Garlic Scape Satay Dip
Community-Tested Pick Candidates (Tested by you!)
Rustic Ramp Tart
R & R Chutney
Sauteed Skate with Melted Ramp Butter
Georgian Khachapuri Filled with Ramp, Green Onions, Herbs and Cheese
The Evasive Calçot and the Promiscuous Romesco
Onion Confit with Honey, Sultanas and White Wine
Wild Green Potatoes
Kutabi, Azerbaijani Savory Pancakes filled with Greens and Herbs
Grilled Cheddar Sandwich with Spring Allium
Pickled Spring Onions
Sake Glazed Salmon with Garlic Chive Egg Noodles
Spring Onion and Lemon Focaccia
Allium Bisque with Frizzled Leeks & Rosemary
Green Goddess Dressing (or Ramp Tramp Dressing)
Garlic Scape Bread
Scallion and Potato stuffed Bread - Alu Parantha
Photos by James Ransom


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Academic and Career Exploration
An MVCC student has the opportunity to explore and develop their goals from the day classes start. Though each student declares a major upon being admitted, students often discover that first choice is no longer the best match. Successful students explore their own interests and develop their goals as career interests change and narrow.
Submitting a request to change majors is the last step of career exploration.
Career Assistance
The MVCC Counseling Center offers CHOICES as a tool for students to explore interests, skills and work values to focus their academic and career goals. The computer-based inventory not only allows the student to explore their personal goals, but also contains a database of job descriptions, requirements, and salaries. An additional feature is a database that links jobs to a searchable database of educational programs throughout the United States and Canada. The Myers-Briggs and Strong Interest Inventory are also available.
The following resources are offered as options and are NOT meant to replace discussions with a Counselor:
Counseling Center
Contact Information
James Maio
Associate Dean
Student Development & Transition Services
Utica Campus
Student Services Center
Phone: (315) 792-5326
Rome Campus
Plumley Complex A30
Phone: (315) 334-7709


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- Quicklook Philips Smart Pasta Maker Sugg. Price $499 Our Price $249.95
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Part of the satisfaction of entertaining guests is making your own unique culinary creations. Treat your friends and family to a dinner of spaghetti bolognese, carbonara or fettucini with noodles you make with electric pasta machines from Williams-Sonoma. Find a stand-alone appliance with the sole purpose of making your favorite pastas or find an attachment that lets you turn an existing appliance into a pasta powerhouse. Keep these appliances next to your other electric cookware for a fully stocked kitchen that rivals any professional chef's.
When you are in a rush to make the perfect batch of pasta noodles, consider using the Philips Smart Pasta maker that produces a pound of product in less than 15 minutes. The appliance comes with eight different discs that help you produce vermicelli, angel hair, lasagna and penne without any additional work. The appliance weighs the amount of flour you add to it, letting you know just how much egg and water you need for the perfect batch. Thanks to its sleek design, you can place the device next to your favorite indoor grills and panini presses for all of your cooking needs.
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If you already have a mixer from a brand such as KitchenAid®, there is no need to invest in a brand new electric pasta machine. You can turn your existing appliance into a super-efficient pasta machine with an attachment, such as the pasta roller or ravioli attachment. Both of these attachments are compatible with all KitchenAid® stand mixers and are easy to install. The stainless-steel construction is durable for those who make a lot of pasta. The pasta roller attachment is designed for making egg noodles, tortellini and lasagna, and the cutter attachment lets you transform sheets into spaghetti and fettuccine with ease. The ravioli attachment separates your pre-made pasta and lets you insert your favorite fillings fresh from the skillet to hand crank into individual dumplings.
While electric pasta machines are perfect for making the main part of the meal, no authentic Italian pasta dish is complete without a great sauce to accompany it. Find a professional-grade food processor to turn solid ingredients into a delicious sauce. Take a bunch of vine-ripened tomatoes and garlic for a classic red sauce, or add cream and cheese to the food processor for a rich white sauce. You can also find dicing food processors that leave the ingredients in a solid state so your guests can experience the exquisite crunch of your homegrown red peppers or zucchini.
Create a true Italian restaurant experience with your electric pasta maker by using toasters and electric ovens to make garlic bread or breadsticks with a crispy crunch. You can even find pizza crispers and ovens that allow you to serve guests a smorgasbord of food for a potluck or a buffet-style dinner.
Complete your meal with a bottle of wine or a nice beer to go along with it. Keep your spirituous beverages at the perfect temperature with a wine and beer fridge. These specially made refrigerators allow you to store several bottles of vino at the perfect temperature for when you are ready to serve them. With special UV-protected glass, the bottles are kept safe from the effects of light. If you have many guests you need to serve in a short amount of time, consider getting a fridge with a built-in tap that lets you serve cold draft beer at a moment's notice.


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Wednesday, March 18, 2009
(Almost) Wordless Wednesday
Edna Maud Pate, Natchitoches, La., c. 1921. Scan of photo privately held by Liz Hall Morgan.
A belated birthday & St. Patrick's Day salute to my maternal grandmother, Edna Maud Pate Stevenson. She was born on St. Paddy's, her favorite color was green, and she died her hair back to its natural red until she died just before her 90th birthday, in 1986. A true (part-)Irish lassie for the ages.
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50 States Song for Kids/50 States and Capitals for Children/USA 50 States
- published: 11 Jan 2016
- views: 12026116
Learn about the 50 states and capitals of the united states of America with this fun educational music video for children and parents. Brought to you by Kids Learning Tube. Don\'t forget to sing along. https://kidslearningtubeshop.com/products/video-membership Watch Kids Learning Tube ad-free for $12 a YEAR! See the latest videos before anyone else in the world. Sign up today for ad-free video streaming for all Kids Learning Tube videos! https://www.patreon.com/kidslearningtube Support Kids Learning Tube by becoming a Patreon today at the link below! You can vote for the video of the week, get your name in the credits and support something you believe in! KLT Website: https://kidslearningtubeshop.com/ T-Shirts: https://kidslearningtubeshop.com/collections/featured-collection Music Downloads: https://kidslearningtubeshop.com/collections/music Facebook: https://www.facebook.com/kidslearningtube Subscribe: http://www.youtube.com/c/kidslearningtube Tweet Us: https://twitter.com/learningtube Instagram: https://instagram.com/kidslearningtube Add us on Google+: https://plus.google.com/+KidsLearningTube iTunes: http://itunes.apple.com/album/id1192890817?ls=1&app=itunes Music: Copyright 2015, 2016, 2017, 2018 Kids Learning Tube Video: Copyright 2015, 2016, 2017, 2018 Kids Learning Tube Lyrics: We're the United States of America We're 50 strong and proud Heres the names of all our states And their capitals sang loud My name is Alabama My capital's Montgomery Welcome to Alaska Juneau's a great place to see I am Arizona My capital is Phoenix My names Arkansas My Li...


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In game one Coker looked to be having a good day as they jumped out to an early 1-0 lead in the second inning, but
Coker held
Knight's pitcher Scott Adams (2-1) earned the win tossing three innings and allowing one run in relief, while Chrusch (7) picked up the save.
For Coker, Steven Dill (2-3) took the loss, going seven and two-thirds innings, allowing eight runs on eight hits, with four strike outs.
For Coker offensively, Nic Fletcher was 3-for-5 with two doubles, a triple and three RBIs, while Ben Davidson was 2-for-4 with a double, an RBI and two runs scored.
The Knights were led by Michael Eastwood, who was 3-for-4 with two RBIs and Anthony Mehling, going 2-for-5 with three RBIs.
Game Two got off to a very bad start for Coker, as
Coker starter Seth Weinberg (2-1) took the loss after throwing two-thirds of an inning, allowing nine runs off eight hits. Kyle Strickland finished the game for the Cobras going six and a third innings scattering nine hits and allowing one run with three strike outs.
As for
Coker's offensive leader was Steven Dill, going 2-for-2 with a run scored, while Willie LoVerde was 1-for-3 with a double.
Coker and


text: This hotel in San Francisco ,California offers every guests, travellers and businessman a comfy place to stay for a night or even for a week,. It has high tech amenities that would surely caught your interest. The staffs are quite accommodating, nice and very welcoming . This is also a perefect place to settle once you seek for a peaceful yet lovely hotel.


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text: Nothing new in programming. In general when using MPI commands even doctors in computer science manage to slow down their software factor 40 first, in order to get better scaling. In computerchess a very normal phenomena. Others manage to get a worse exponent even, like Deep Blue did, using wrong algorithms. At 10:01 AM 4/26/2005 -0500, Ben Mayer wrote: >Just a quick comment about CELL and gcc and vectorizing. > >I looked over the specs of CELL and it looks very much like a hybrid >vector processor. gcc now has support for vectorizing (some) loops. > >I spend a good deal of time writing code for Cray X1Es (vector >supercomputer), and there are a lot of things that can trip up a >compiler when one is trying to get it to vectorize code. I actually >just did a small study of how well students in a parallel computing >class write parallel codes on X1 with MPI and UPC. One of the things >that stood out is that they tended to do odd things in their loops >that inhibit code from vectorizing. > >Writing code which vectorizes well is difficult. The reason I mention >this is that, I believe, we will see that someone *has to* produce >libraries (or entire engines) that take advantage of the Cells unique >layout to get decent performance. If there are major parts of the code >which can not run in parallel (Vectorization is a type of parallel >execution) Cell is just a crummy PPC chip. > >Ben > >_______________________________________________ >Beowulf mailing list, Beowulf at beowulf.org >To change your subscription (digest mode or unsubscribe) visit http://www.beowulf.org/mailman/listinfo/beowulf > >


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|HIV & the A.C.A.
Oct 2, 2013
I get my medical bills paid for by Charity Care for the Hospital I go to (St. Mary's, Passaic, NJ) and my meds by P.A.A.D. How will the A.C.A. affect the coverage I have. I grow less than 16k per year, and am 58 years old. Will I have to join or buy into insurance exchanges? Will my prescriptions still be covered without a deductible by me?
Response from Mr. Chambers
Your question is perfectly timed as the enrollment into ACA (Affordable Care Act aka Obamacare) just opened. There has been a lot of concern about how Ryan White funded plans are going to coordinate with the new law.
To answer your questions, I am assuming that the Charity Care you receive is for basic HIV care. You should ask them to be sure, but I doubt if that counts as a qualified health insurance plan that you will need to have to avoid a tax penalty next year. If that is the case you will need to enroll in a health plan.
Based on your income, you will either be eligible for Medicaid or, more likely, you will need to purchase a health plan from the Health Exchange.
At your income level, the federal government will pay a large portion of your premiums. Also New Jersey has a Health Insurance Continuation Program (HICP) through their state office of AIDS, which will probably pay your portion of any premiums.
Your health exchange is located here. Just enter your state and it will show you plans, what your costs would be based on your income.
I'm sorry I can't be more definite about this, but since federal Ryan White funds for HIV care is administered by the states, there may be slight differences in the programs by state. I encourage you to contact a local AIDS Service Organization in your area. Someone there should be able to give you more specific information.
Regarding medications, when you say P.A.A.D. I wonder if you mean the AIDS Drug Distribution Program (A.D.D.P.) That is the program in New Jersey that pays for HIV medications. That program will continue.
When you purchase a health insurance plan under ACA, the ADDP program will pay what the insurance does not pay for medications that are on the ADDP formulary, so, no, you will not have a deductible for the medications you get through ADDP. If I am mistaken about the program, please write back.
To summarize, check your health exchange and see what you are eligible for based on your income. Contact a local AIDS Service Organization to see how the programs that are unique to PLWHA in New Jersey will help you with ACA coverage.
This is a whole new era for health insurance in this country so please be patient. There will be glitches and bumps in the initial process, but millions more are going to have access to quality health care with this health insurance. Good luck, Jacques located here.
sore throat
Federal background checks for employment
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