Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5–15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery

<i>Firmicutes</i> to <i>Bacteroidetes</i> ratio.

<p><i>Firmicutes</i> to <i>Bacteroidetes</i> ratio in the colon contents (lumen) was significantly higher (ANOVA p = 0.0021, post-tests both p<0.01) in the traditional diet fed group treated with antibiotics compared with either defined nutrient diet with antibiotics.</p

Effects of nutritional status and diets on mortality, disease severity and infection burden in mice challenged with <i>C</i>. <i>difficile</i>.

<p>Effects of nutritional status and diets on mortality, disease severity and infection burden in mice challenged with <i>C</i>. <i>difficile</i>.</p

Defined Nutrient Diets Alter Susceptibility to <i>Clostridium difficile</i> Associated Disease in a Murine Model

<div><p>Background</p><p><i>Clostridium difficile</i> is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of <i>C</i>. <i>difficile</i> infection in a murine model.</p><p>Methods</p><p>C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with <i>C</i>. <i>difficile</i> strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and <i>C</i>. <i>difficile </i>growth and toxin production were quantified <i>ex vivo </i>in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.</p><p>Results</p><p><i>C</i>. <i>difficile </i>infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. <i>C</i>. <i>difficile</i> shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. <i>Ex vivo</i> inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and <i>C</i>. <i>difficile</i> growth in both defined diets compared with a traditional diet.</p><p>Conclusions</p><p>Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.</p></div

Treatment schedule for mice for the <i>in vivo</i> infection model (A) and <i>ex vivo C</i>. <i>difficile</i> inoculation studies (B).

<p>Treatment schedule for mice for the <i>in vivo</i> infection model (A) and <i>ex vivo C</i>. <i>difficile</i> inoculation studies (B).</p

<i>C</i>. <i>parvum</i> priming enhances Th1-type cytokine responses to re-challenge in protein malnourished mice.

<p>(A) Ileal inflammatory mediators and chemokines and (B) cytokines measured three days after 10⁷ <i>C</i>. <i>parvum</i> challenge in previously uninfected (PBS) compared with mice primed with 10⁶ <i>C</i>. <i>parvum</i> (Cp10^<i>6</i>) 20 days prior to re-challenge. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>C</i>. <i>parvum</i> priming leads to sustained changes in ileal tissue chemokine and cytokine profiles during protein malnutrition.

<p>Mice were conditioned on PD for 5 days prior to infection with 10⁶ <i>C</i>. <i>parvum</i> (Cp10⁶). Luminex was performed for measurement of chemokines and cytokines in ileal tissues at. day 3 (D3) and day 23 (D23) post challenge compared to uninfected controls (PBS) (n = 3-4/group). (A) Primary <i>C</i>. <i>parvum</i> infection led to increases in CXCL9, CXCL10, CCL-3, CCL-5, and CCL11 on D3. On D23, TNFα, IL1β, and IL-8 were diminished in infected mice relative to uninfected controls, however, CCL-5 continued to be elevated and other chemokines had returned to baseline. (B) Only IL12p40 and IL-13 were modestly elevated three days after primary <i>C</i>. <i>parvum</i> challenge. There was a relative decrease in all Th2-type cytokines through 23 days post-<i>C</i>. <i>parvum</i> compared with uninfected controls. (n = 3-4/group). *<i>P</i><0.05 for PBS vs Cp10⁶ as indicated; #<i>P</i><0.05 for Cp10⁶ D3 vs Cp10⁶ D23 as indicated.</p

Protein malnutrition alters basal immune responses to primary <i>C</i>. <i>parvum</i> exposure, but secondary responses are intact.

<p>Immunologic responses to two different recombinant <i>Cryptosporidium</i> sporozoite antigens (CApy and Cp15) were performed at 13–15 days post <i>C</i>. <i>parvum</i> challenge in mice fed either control-diet (<i>C</i>. <i>parvum</i>^CD) or protein-deficient diet (<i>C</i>. <i>parvum</i>^pd) and results were compared with naïve age and diet-matched uninfected controls (PBS^CD and PBS^pd). Mice began respective diets 12 days prior to <i>C</i>. <i>parvum</i> challenge and remained on the same diets post-challenge. (A) Cytokine secretion in splenocytes of naïve (uninfected) CD or PD-fed mic after stimulation with <i>Cryptosporidium</i> antigens. (B) Serum antibody production as anti-CApy or anti-Cp15 IgG titer (<i>*P<</i>0.05). (C) Cytokines secreted after CApy or Cp15 antigen stimulation in (C) mesenteric lymph nodes. (D) Cytokine secretion in splenocytes expressed as fold change relative to CD-fed uninfected controls. (*<i>P</i><0.05 as indicated). Data is representative of pooled individual responses from two separate tissue harvests (n = 4-5/group).</p

Severe protein malnutrition in mice selectively enhances intestinal disruption and severity of cryptosporidiosis.

<p>(A) Experimental timeline. 3-week-old C57Bl/6 female mice were initiated on experimental malnutrition diets (RBD or PD) or control diet (CD) immediately upon receipt from supplier. Challenge with 10⁶ or 10⁷ <i>Cryptosporidium parvum</i> oocysts, heat-inactivated <i>C</i>. <i>parvum</i> oocysts (Δ<i>C</i>. <i>parvum</i>) or PBS occurred via oral gavage 5 days after initiating diet. Serial weights were collected daily post-challenge, and fecal parasite shedding was determined by RT-PCR. On day 3–4 post-challenge, tissue parasite burden and mucosal injury was assessed by measuring ileum villus:crypt ratios and alterations in epithelial tight-junction proteins. (B) Impact of diet on growth (**<i>P</i><0.01, ***<i>P</i><0.001 <i>C</i>. <i>parvum</i>^PD vs. <i>C</i>. <i>parvum</i> or <i>C</i>. <i>parvum</i>^RBD), fecal parasite shedding (**<i>P</i><0.01 PD or RBD vs. CD day 2, *<i>P</i><0.05 PD vs. RBD or CD day 3), tissue parasite burden (*<i>P</i><0.05 PD vs CD or RBD ileum, *<i>P</i><0.05 PD vs CD colon), and ileum villus:crypt ratios (**<i>P</i><0.01 PBS^CD vs PBS^RBD, ***<i>P</i><0.001 PBS^CD vs PBS^PD, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i>^CD or <i>C</i>. <i>parvum</i>^RBD vs <i>C</i>. <i>parvum</i>^PD, ^<i>P</i><0.05 PBS^CD vs. <i>C</i>. <i>parvum</i>^CD, ^##<i>P</i><0.01 PBS^PD vs. <i>C</i>. <i>parvum</i>^PD (n = 3-7/group). (C) Growth through three days post-challenge with either <i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum</i>. (N = 5-10/group, **<i>P</i><0.01, ***<i>P</i><0.001 (<i>C</i>. <i>parvum</i> vs. either PBS or Δ<i>C</i>. <i>Parvum</i>) and fecal parasite shedding (n = 5-10/group, *<i>P</i><0.05, **<i>P</i><0.01). (D) Immunofluorescence staining of epithelial cell tight-junction proteins (ZO-1, occludin, claudin-2) in ileum of CD and PD-fed infected mice and uninfected controls (n = 4/group). (E) Dose dependent persistent growth faltering (n = 10/group, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i> 10⁷ vs <i>C</i>. <i>parvum</i> 10⁶; and ^####<i>P</i><0.0001 <i>C</i>. <i>parvum</i> vs PBS) and (F) fecal parasite shedding (**<i>P</i><0.01, ****<i>P</i><0.0001) through 21 days post-challenge. Data is representative of 2 replicate experiments.</p

Viable <i>C</i>. <i>parvum</i> priming provides greater protection against re-challenge than either CpG-ODN or <i>S</i>. Typhi.

<p>(A, B) Comparison of protective immunity following priming with either viable and heat-inactivated (Δ) <i>C</i>. <i>parvum</i> 10⁶. (A) Growth of PD-fed mice through 23 days post-priming with either 4x10⁶ viable (<i>C</i>. <i>parvum</i>) or 4x10⁶ heat-inactivated (Δ<i>C</i>. <i>parvum</i>). Mice were challenged with viable 4<i>x</i>10⁷ <i>C</i>. <i>parvum</i> oocysts on day 20 post-priming. *<i>P</i><0.05 for Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i> vs. <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> (d3 and d23); ^^<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-PBS vs PBS-<i>C</i>. <i>parvum</i> (d23); ^###<i>P</i><0.001 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs. PBS-<i>C</i>. <i>parvum</i> (d23). (B) RT-PCR of <i>Cryptosporidium</i> stool shedding on experimental days 21 and 23 (day 1 and day 3 after <i>C</i>. <i>parvum</i> 10⁷ challenge, respectively). *<i>P</i><0.05 and *<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs either PBS-<i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i>. (C,D) 3-week-old C57Bl/6 mice were conditioned on PD for 7 days prior to orogastric inoculation with 10⁶ <i>C</i>. <i>parvum</i>, intranasal (i.n.) 10⁹ <i>S</i>. Typhi 908<i>htr</i>, i.n. CpG-ODN 1668 (100 mcg), or PBS (100 mcl) as indicated (n = 10/group). On day 21, mice were re-challenged with either PBS or <i>C</i>. <i>parvum</i> 10⁷. (C) Growth as percentage of initial weight, normalized to the day of 10⁷ <i>C</i>. <i>parvum</i> challenge (Day 0). The group labeled “All uninfected” includes animals that received either PBS during both inoculations, CpG followed by PBS, or <i>S</i>. Typhi followed by PBS (n = 5/group x 3 = 15) given all three groups grew similarly and were never exposed to <i>C</i>. <i>parvum</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004820#pntd.0004820.s004" target="_blank">S4 Fig</a>). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 for PBS–<i>C</i>.<i>p</i>.10⁷ (red) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ (yellow) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷). Horizontal lines designate significant differences at <i>P</i><0.05 between CpG-<i>C</i>.<i>p</i>.10⁷ (yellow), <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green), and PBS-<i>C</i>.<i>p</i>.10⁷ (red) vs. All uninfected controls, respectively. (D) Parasite fecal shedding in serial fecal pellets collected on indicated experimental days post <i>C</i>. <i>parvum</i> 10⁷ challenge. *<i>P</i><0.05 for PBS–<i>C</i>.<i>p</i>.10⁷ vs. <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷. Data is representative of two replicate experiments.</p