Lopa M. Das

TGF-β conditions intestinal T cells to express increased levels of miR-155, associated with down-regulation of IL-2 and itk mRNA

Transforming growth factor (TGF)-β, is an immunosuppressive cytokine that inhibits T-cell activation. We hypothesized that TGF-β mediates its immunoinhibitory effects by modulation of micro RNA (miRNA)-155 (miR-155). Interleukin (IL)-2 and interferon-γ are down-regulated by TGF-β in activated CD4 peripheral blood T cells and lamina propria T cells (LPT), but miR-155 is upregulated ninefold specifically in LPT. Consequently, this study focuses on the role of TGF-β-enhanced miR-155 on LPT immune responses. TGF-β induces miR-155 in both freshly isolated and LPT lymphoblasts, whereas other inducible miRNAs are not regulated by TGF-β. Using MAMI bioinformatics database, we determined that inducible T-cell kinase (itk) is a functional target of miR-155 that exhibits an inverse mRNA response to that of miR-155. To determine experimentally that miR-155 regulates itk, transfection experiments were performed that demonstrated miR-155 overexpression decreased itk and IL-2 mRNA, whereas antagonism of miR-155 restored both mRNAs in activated cells. These findings describe a TGF-β-dependent function for miR-155 in modulating cytokine and T-cell immune responses in the gut.

Suppression of Hyperactive Immune Responses Protects against Nitrogen Mustard Injury

DNA alkylating agents like nitrogen mustard (NM) are easily absorbed through the skin and exposure to such agents manifest not only in direct cellular death but also in triggering inflammation. We show that toxicity resulting from topical mustard exposure is mediated in part by initiating exaggerated host innate immune responses. Using an experimental model of skin exposure to NM we observe activation of inflammatory dermal macrophages that exacerbate local tissue damage in an inducible nitric oxide synthase (iNOS)-dependent manner. Subsequently these activated dermal macrophages reappear in the bone marrow to aid in disruption of hematopoiesis and contribute ultimately to mortality in an experimental mouse model of topical NM exposure. Intervention with a single dose of 25-hydroxyvitamin D3 (25(OH)D) is capable of suppressing macrophage-mediated iNOS production resulting in mitigation of local skin destruction, enhanced tissue repair, protection from marrow depletion, and rescue from severe precipitous wasting. These protective effects are recapitulated experimentally using pharmacological inhibitors of iNOS or by compounds that locally deplete skin macrophages. Taken together, these data highlight a critical unappreciated role of the host innate immune system in exacerbating injury following exposure to NM and support the translation of 25(OH)D in the therapeutic use against these chemical agents.

Hyper-Inflammation and Skin Destruction Mediated by Rosiglitazone Activation of Macrophages in IL-6 Deficiency

Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation–activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency.

Defining the timing of 25(OH)D rescue following nitrogen mustard exposure

Abstract Objective: Mass exposure to alkylating agents such as nitrogen mustard (NM), whether accidental or intentional as during warfare, are known to cause systemic toxicity and severe blistering from cutaneous exposure. Thus, establishing the timing and appropriate dose of any potential drug designed to reverse or impede these toxicities is critical for wound repair and survival. Our previous data demonstrates that a single intraperitoneal injection of low-dose 25-hydroxyvitamin D3 (25(OH)D) given as early as 1 h following NM exposure is sufficient to rescue mice from pancytopenia and death. However, the duration of time following exposure where intervention is still effective as a countermeasure is unknown. In this study, we sought to assess the maximal time permissible following NM exposure where 25(OH)D still affords protection against NM-induced cutaneous injury. Additionally, we determined if a higher dose of 25(OH)D would be more efficacious at time interval where low dose 25(OH)D is no longer effective. Methods: Low (5 ng) and high (50 ng) doses of 25(OH)D were administered intraperitoneally to mice following exposure to topical NM to assess wound resolution and survival. Mice were imaged and weighed daily to measure wound healing and to monitor systemic toxicity. Results: We demonstrated that 5 ng 25(OH)D administered as early as 1 h and as late as 24 h post-NM exposure is able to achieve 100% recovery in mice. In contrast, intervention at and beyond 48 h of NM exposure failed to achieve full recovery and resulted in ≥60% death between days 6 and 12, demonstrating the critical nature of timely intervention with 25(OH)D at each respective dose. In order to circumvent the observed failure at >48 h exposure, we provided two consecutive doses of 5 ng or 50 ng of 25(OH)D at 48 h and 72 h post-NM exposure. Repeat dosing with 25(OH)D at 48 h and beyond led to marked improvement of lesion size with 75% recovery from mortality. Conclusions: The opportunity to use 25(OH)D as a medical countermeasure for NM-induced toxicity has a finite of window for intervention. However, modifications such as repeat dosing can be an effective strategy to extend the intervention potential of 25(OH)D.

Early indicators of survival following exposure to mustard gas: Protective role of 25(OH)D

The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25 (OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25 (OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure.

The Virtues of Oxygenation: Low Tissue Oxygen Adversely Affects the Killing of Leishmania

Hypoxia contributes to the persistence of infections through altered immune responses. Studies examining skin O2 changes at the site of a lesion are limited. The prevailing methods require the use of electrochemical O2 sensors or radiolabeled electrodes that utilize O2 and may interfere with the precision at low O2 levels. In this issue, Mahnke et al. (2014) demonstrate, using a novel fluorescence-based imaging technology, that low oxygen tension (pO2) impairs NO-mediated anti-leishmanial immunity, leading to increased parasite burden. Replenishing tissue oxygen profoundly enhanced NO-mediated leishmanial killing, underscoring the need to accurately assess oxygenation in infected tissues as a novel strategy to challenge intracellular infection. The technology presented here may have clinical-translational potential in noninvasively assessing disease burden and response to treatment.