Modeling Asthma in Mice Using Common Aeroallergens.

Aeroallergens are common inducers of asthma in humans and are widely used in experimental research to generate animal models of this disease. In this chapter, we describe four mouse models of aeroallergen-induced asthma. These models differ in type and number of allergens used, route and duration of allergen exposure, and utilization of an adjuvant, representing different mechanistic variants of asthma. In addition, we describe several basic methods that are commonly used in mechanistic studies of asthma in mice. These methods include tracheotomy and bronchoalveolar lavage, cytospin and morphologic analysis of bronchoalveolar lavage cells, and lung harvest and digestion for generation of single-cell suspension.

Toll-Like Receptor 2 Targeted Rectification of Impaired CD8⁺ T Cell Functions in Experimental Leishmania donovani Infection Reinstates Host Protection.

Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.

Glycyrrhizic acid-mediated subdual of myeloid-derived suppressor cells induces antileishmanial immune responses in a susceptible host.

CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), a heterogeneous population of precursor cells, modulate protective immunity against visceral leishmaniasis by suppressing T cell functions. We observed that CD11b(+) Gr1(+) MDSCs, which initially expanded in soluble leishmanial antigen (SLA)-immunized mice and later diminished, suppressed proliferation of T cells isolated from SLA-immunized mice, but to a lesser extent than the case in naive mice. This lesser suppression of MDSCs accompanied the expression of F4/80 and the production of Cox-2, arginase I, nitric oxide, and PGE2. However, with SLA immunization, there was no difference in the expression of interleukin-2 (IL-2) or gamma interferon (IFN-γ) by T cells, in contrast to the case in nonimmunized mice, in which there is an influence. Glycyrrhizic acid (a triterpenoid compound)-mediated inhibition of Cox-2 in myeloid-derived suppressor cells influenced the capacity of T cells to proliferate and the expression of IL-2 and IFN-γ in Leishmania donovani-infected BALB/c mice. Further characterization confirmed that administration of glycyrrhizic acid to L. donovani-infected BALB/c mice results in an impairment of the generation of MDSCs and a reciprocal organ-specific proliferation of IFN-γ- and IL-10-expressing CD4(+) and CD8(+) T cells. Comprehensive knowledge on the Cox-2-mediated regulation of myeloid-derived suppressor cells might be involved in unlocking a new avenue for therapeutic interventions during visceral leishmaniasis.

Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

Antigen-Pulsed CpG-ODN-Activated Dendritic Cells Induce Host-Protective Immune Response by Regulating the T Regulatory Cell Functioning in Leishmania donovani-Infected Mice: Critical Role of CXCL10.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a systemic infection of reticulo-endothelial system. There is currently no protective vaccine against VL and chemotherapy is increasingly limited due to appearance of drug resistance to first line drugs such as antimonials and amphotericin B. In the present study, by using a murine model of leishmaniasis we evaluated the function played by soluble leishmanial antigen (SLA)-pulsed CpG-ODN-stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular parasitic growth. We establish that a single dose of SLA-CpG-DC vaccination is sufficient in rendering complete protection against L. donovani infection. In probing the possible mechanism, we observe that SLA-CpG-DCs vaccination results in the significant decrease in Foxp3(+)GITR(+)CTLA4(+)CD4(+)CD25(+) regulatory T cells (Treg) cell population in Leishmania-infected mice. Vaccination with these antigen-stimulated dendritic cells results in the decrease in the secretion of TGF-ß by these Treg cells by possible regulation of the SMAD signaling. Moreover, we demonstrate that a CXC chemokine, IFN-γ-inducible protein 10 (IP-10; CXCL10), has a direct role in the regulation of CD4(+)CD25(+) Treg cells in SLA-CpG-DC-vaccinated parasitized mice as Treg cells isolated from IP-10-depleted vaccinated mice showed significantly increased TGF-ß production and suppressive activity.

CXCL10 is critical for the generation of protective CD8 T cell response induced by antigen pulsed CpG-ODN activated dendritic cells.

The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8⁺ T cell-mediated immune response. We observed significant reduction of CD8⁺ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8⁺ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8⁺ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against Leishmania donovani infection.