Behaviorally mediated, warm adaptation : a physiological strategy when mice behaviorally thermoregulate

Laboratory mice housed under standard vivarium conditions with an ambient temperature (T-a) of similar to 22 degrees C are likely to be cold stressed because this T-a is below their thermoneutral zone (TNZ). Mice raised at T(a)s within the TNZ adapt to the warmer temperatures, developing smaller internal organs and longer tails compared to mice raised at 22 degrees C. Since mice prefer T(a)s equal to their TNZ when housed in a thermocline, we hypothesized that mice reared for long periods (e.g., months) in a thermocline would undergo significant changes in organ development and tail length as a result of their thermoregulatory behavior. Groups of three female BALB/c mice at an age of 37 days were housed together in a thermocline consisting of a 90 cm long aluminum runway with a floor temperature ranging from 23 to 39 degrees C. Two side-by-side thermoclines allowed for a total of 6 mice to be tested simultaneously. Control mice were tested in isothermal runways maintained at a T-a of 22 degrees C. All groups were given cotton pads for bedding/nest building. Mass of heart, lung, liver, kidney, brain, and tail length were assessed after 73 days of treatment. Mice in the thermocline and control (isothermal) runways were compared to cage control mice housed 3/cage with bedding under standard vivarium conditions. Mice in the thermocline generally remained in the warm end throughout the daytime with little evidence of nest building, suggesting a state of thermal comfort. Mice in the isothermal runway built elaborate nests and huddled together in the daytime. Mice housed in the thermocline had significantly smaller livers and kidneys and an increase in tail length compared to mice in the isothermal runway as well as when compared to the cage controls. These patterns of organ growth and tail length of mice in the thermocline are akin to warm adaptation. Thus, thermoregulatory behavior altered organ development, a process we term behaviorally mediated, warm adaptation. Moreover, the data suggest that the standard vivarium conditions are likely a cold stress that alters normal organ development relative to mice allowed to select their thermal preferendum.Foundation for Polish Science European Commission (MPD/2009-3/5/stp)European CommissionFundacja na rzecz Nauki Polskie

Stressful Presentations: Mild Chronic Cold Stress in Mice Influences Baseline Properties of Dendritic Cells

The ability of dendritic cells to stimulate and regulate T cells is critical to effective anti-tumor immunity. Therefore, it is important to fully recognize any inherent factors which may influence DC function under experimental conditions, especially in laboratory mice since they are used so heavily to study immune responses. Physiological stress is well recognized to impair several arms of immune protection. The goals of this report are to briefly summarize previous work revealing how DCs respond to various forms of physiologically relevant stress and to present new data highlighting the potential for chronic mild cold stress inherent in mice housed at standard ambient temperatures required for laboratory mice to influence baseline DCs properties. Since recent data from our group shows that CD8+ T cell function is altered by mild chronic cold stress and since DC function is crucial for CD8+ T cell activation, we wondered whether mild cold stress may also be influencing DC properties. We found increased numbers of splenic DCs (CD11c+) in cold stressed mice compared to mice housed at a thermoneutral temperature, which significantly reduces cold stress. However, many of the DCs which are expanded in cold stressed mice express an immature phenotype. We also found that antigen presentation and ability of splenocytes to activate T cells were impaired compared to that seen in DCs isolated from mice at thermoneutrality. The new data presented here strongly suggest that the housing temperature of mice can affect fundamental properties of DC function which in turn could be influencing the response of DCs to added experimental stressors or other treatments

Defining immunological impact and therapeutic benefit of mild heating in a murine model of arthritis.

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as "alternative therapies". In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA

In vitro heat treatment inhibits LPS-induced cytokine production by activated macrophages.

<p>(A-B), BALB/c mice were injected intraperitoneally with 10 μg of LPS. Peritoneal macrophages were harvested 3 days post LPS injection, recovered overnight and re-stimulated (2x10⁵/well) with LPS (100 ng/mL) and IFN-γ (25 U) at 37°C or 39.5°C for 6 hours to determine TNF-α, IL-6, IL-1β and IL-10 production by ELISA (A) or re-stimulated (1x10⁶/well) for 4 hours to measure TNF-α, IL-6, IL-1β and IL-10 mRNA expression by quantitative real-time PCR (B). The results are presented relative to GAPDH and baseline expression in unstimulated cells at 37°C. Cells from each treatment condition were pooled from 2–4 mice and measured in triplicate. Data are mean ± SD. Data are representative of three independent experiments. * p < 0.05, paired Student <i>t</i> test and repeated-measures two-way ANOVA.</p

Multiple targets are affected by heat treatment.

<p>(A), Serum TNF-α concentration from CIA mice was determined by ELISA. Error bars show SEM (n = 7). (B-C), TNF-α and IL-10 concentrations were detected in the tissue homogenates from naïve and CIA mice paws by ELISA (day 53). Error bars show SEM. Data are representative of two experiments. (D-E), tissue homogenates were prepared from naïve and CIA mice paws and expression of phosphorylated IKKα /β, NF-κB p65, p50, HSP70 and HIF-1α were detected by Western blotting. Each lane represents different mice. The graph shows the ratio of the band intensity of proteins normalized to β-actin. * p < 0.05, paired Student <i>t</i> test to compare treated to untreated group.</p

Heat treatment reduces arthritis disease severity and joint damage in a collagen-induced arthritis mouse model.

<p>(A-C), DBA1 mice were immunized with bovine CII and either received HT (6 hrs, 2x/week) or left untreated for total 9 weeks, beginning on day 22 (arrow). Data are presented as (A) incidence of arthritis in the indicated groups (n = 8) and (B) mean disease severity scores ± SEM (n = 7). (C), H&E-stained section of synovial toe joints from representative naïve and arthritic mice (day 88). Synovial hyperplasia and immune cell infiltration was shown by the arrow. B: bone; *: joint space. Scale bar, 100 μm. (D), For the prophylactic study, mice received HT from day 22, whereas in the therapeutic study, mice received HT starting at day 35 when the mean disease severity score reached 2. * p < 0.05, non-parametric Mann-Whitney U-test to compare treated to untreated mice. (E), Flow cytometric analysis of CD11b⁺ macrophage infiltration in the joints of naïve and CIA mice (day 88) (n = 4 for naïve and n = 9 for CIA mice). (F), LN cells were restimulated with bovine CII (0, 50, 100 μg/ml) for 72 hr. Supernatants were collected to determine the levels of IFN-γ by ELISA. Data shows individual mice. * p < 0.05, paired Student <i>t</i> test (G), Serum levels of anti-CII IgG, IgG1, IgG2a and IgG2b were detected by ELISA (n = 8). Error bars show SEM. Data are representative of three experiments. * p < 0.05, paired Student <i>t</i> test or repeated-measures two-way ANOVA to compare treated to untreated group.</p

Effects of heat treatment and combination therapy with methotrexate in controlling the development of arthritis.

<p>(A-C), DBA1 mice were immunized with bovine CII and received HT (6 hrs, 2x/week), MTX (5 mg/kg, 3x/week), HT in combination with MTX or left untreated for total 5 weeks, beginning on day 22 (arrow). (A), Data are presented as mean disease severity scores ± SEM (n = 5). (B), H&E-stained section of synovial joints from arthritic mice with different treatments (day 53). Synovial hyperplasia and immune cell infiltration was shown by the arrows. (C), Flow cytometric analysis of CD11b⁺ macrophage infiltration in the joints of CIA mice with different treatments (day 53). (D), DBA1 mice were immunized with bovine CII and received short daily 30-minute HT, MTX (5 mg/kg, 3x/week) or HT in combination with MTX from day 22. Data are presented as mean disease severity scores ± SEM (n = 5). (E), Flow cytometric analysis of CD11b⁺ macrophage infiltration in the joints. * p < 0.05, non-parametric Mann-Whitney U-test (A and D) and paired Student <i>t</i> test (C and E) to compare treated to untreated group.</p

<i>In vitro</i> heat treatment increases HSF-1 and HSP 70 expression which may regulate TNF-α production in activated macrophages.

<p>Peritoneal macrophages were harvested from LPS-challenged mice, recovered overnight and re-stimulated with LPS (100 ng/mL) and IFN-γ (25 U) at 37°C or 39.5°C for indicated times. Cell lysates were prepared from these cells to detect HSF-1 and HSP70 by Western blotting. Cells stimulated at 42°C for 30 min were used as positive controls. The graph shows the ratio of the band intensity normalized to β-actin. Data are mean ± SEM. Data are representative of two independent experiments. * <i>p</i> < 0.05, paired Student <i>t</i> test.</p

Effects of <i>In vitro</i> heat treatment on macrophage NF-κB and STAT1 activation.

<p>(A-B), Peritoneal macrophages were isolated from LPS-challenged mice, recovered overnight and re-stimulated (1x10⁶/well) with LPS/IFN-γ at 37°C or 39.5°C for 30 min. (A) These cells were then stained with antibodies against CD11b, NF-κB p65 and DRAQ5 DNA dye and then analyzed by ImageStream flow cytometry. CD11b⁺ cells were gated to show the SS between DRAQ5 nuclear staining and NF-κB staining or (B) nuclear and cytosol proteins were isolated to detect NF-κB p65 expression by Western blotting. The graph shows the ratio of the band intensity of NF-κB p65 normalized to β-actin or PCNA. Data are representative of two independent experiments. (C) Macrophages were stimulated at 37°C or 39.5°C for 1 hour. Cross-linked chromatin was immunoprecipitated with anti-NF-κB p65 antibody and analyzed for NF-κB p65 binding to the TNF-α promoter region by quantitative real-time PCR with primers spanning the regions-364/-182, -586/-468, -685/-543 and-912/-763. Fold change is normalized to the input and control IgG and then compared with unstimulated cells at 37°C. Cells from each treatment condition were pooled from 4 mice. Data are representative of two independent experiments. (D-E), Macrophages were re-stimulated with LPS/IFN-γ at 37°C or 39.5°C for indicated times. (D) Cells were stained with CD11b and IFN-γ receptor antibodies. CD11b⁺ cells were analyzed for expression of IFN-γ receptor by flow cytometry. (E) Cell lysates were prepared to detect phosphorylated STAT1 and total STAT1 by Western blotting. Quantification of the band intensity of pSTAT1 and STAT1 is normalized to β-actin. Cells from each treatment condition were pooled from 4 mice. Data are representative of two independent experiments.</p