CD14 contributes to pulmonary inflammation and mortality during murine tuberculosis

Toll-like receptors play an essential role in the innate recognition of micro-organisms by the host. CD14 is one of the extracellular adaptor proteins required for recognition of Gram-negative bacteria and possibly also Mycobacterium tuberculosis. Therefore, we intranasally infected wild-type (WT) and CD14 knock-out (KO) mice with virulent M. tuberculosis H37Rv. We found no differences in bacterial load in the main target organ lung up to 32 weeks after infection. From 20 weeks onward 57% of WT mice succumbed, whereas all CD14 KO mice survived. The improved outcome of CD14 KO mice was accompanied by reduced pulmonary inflammation; lung cell counts and percentage of inflamed lung tissue were reduced in CD14 WT mice. These data suggest that during chronic infection CD14 KO mice are protected from lethality caused by lung tuberculosis because of a reduction of the inflammatory response.     

Blockade of endogenous leukotrienes exacerbates pulmonary histoplasmosis

Leukotrienes are classical mediators of inflammatory response. New aspects of leukotriene function have recently been described. We examine here the previously unreported role that leukotrienes play in the regulation of cytokines in a murine model of histoplasmosis. We demonstrate that administration of MK 886, a leukotriene synthesis inhibitor, caused Histoplasma capsulatum-infected mice to die by the day 15 of infection, whereas the correlating death rate in untreated infected mice was 0%. Treating infected animals with MK 886 inhibited leukotriene synthesis but increased leukocyte recruitment to the lungs. Subsequent to this phenomenon, levels of tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, and KC chemoattractant cytokines and fungi in the lung parenchyma increased, as did inflammatory response. In contrast, IL-2, IL-5, IL-12, and gamma interferon cytokine levels actually decreased. Thus, murine response to pulmonary histoplasmosis may be leukotriene modulated. This finding may enable us to alter the course of the immune response and inflammation caused by histoplasmosis. The data from the present study suggest an important new strategy for immunologic or drug intervention in human patients.     

Levamisole exacerbates coxsackievirus B3-induced murine myocarditis.

Levamisole administration to several strains of adolescent mice at the time of or up to 4 days post-inoculation (p.i.) with a myocarditic variant of coxsackievirus B3 (CVB3m) increased the number of myocarditic lesions above that found in CVB3m-inoculated mice. Virus replication in heart tissues in vivo was not affected by levamisole administration to the mice, nor was production of neutralizing antibody to CVB3m. Lymphocytes from nodes of virus-inoculated mice treated with levamisole at 2 days p.i. exhibited an increased reactivity to phytohemagglutinin on days 6 and 8 p.i., compared with respective responses by nodal T lymphocytes from CVB3m-inoculated mice. Levamisole treatment of CVB3m-inoculated mice also increased the reactivity of splenic and peripheral blood T lymphocytes to phytohemagglutinin on day 8 p.i., but not day 6 p.i., compared with the respective responses by lymphocytes from CVB3m-inoculated mice. The proportion of theta antigen-bearing lymphocytes in the total lymphocyte population in peripheral blood of CVB3m-inoculated mice was not altered by levamisole treatment. However, CVB3m-induced reduction in this subpopulation of lymphocytes in the nodes was restored to control levels by levamisole treatment. Reactivities of cytotoxic T lymphocytes from CVB3m-inoculated mice were increased against both normal and CVB3m-inoculated target cells after levamisole treatment of these mice. The results suggest that levamisole may contribute to CVB3m induction of myocarditis by several mechanisms, such as increasing the blastogenic activity of the phytohemagglutinin-responding subset of T lymphocytes, by possibly altering T-lymphocyte distribution in the body and by nonspecifically increasing reactivities of cytotoxic T lymphocytes.     

Role of chemokine ligand 2 in the protective response to early murine pulmonary tuberculosis

Chemokines play an important role in the development of immunity to tuberculosis. Chemokine ligand 2 (CCL2, JE, monocyte chemoattractant protein-1) is thought to be primarily responsible for recruiting monocytes, dendritic cells, natural killer cells and activated T cells, all of which play critical roles in the effective control of tuberculosis infection in mice. We show here that in mice in which the CCL2 gene was disrupted, low-dose aerosol infection with Mycobacterium tuberculosis resulted in fewer macrophages entering the lungs, but only a minor and transient increase in bacterial load in the lungs; these mice were still able to establish a state of chronic disease. Such animals showed similar numbers of activated T cells as wild-type mice, as determined by their expression of the CD44hi CD62lo phenotype, but a transient reduction in cells secreting interferon-gamma. These data indicate that the primary deficiency in mice unable to produce CCL2 is a transient failure to focus antigen-specific T lymphocytes into the infected lung, whereas other elements of the acquired host response are compensated for by different ligands interacting with the chemokine receptor CCR2.     

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl-Neelsen staining. CD11c(+) DC and CD14(+) cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII(+) cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14-21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24-48 h). Regarding granuloma constitution, highly bacillary CD14(+) and SRA(+) cells were centrally located. MHC-CII(+) cells were more peripheral, with less mycobacteria. CD11c(+) cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl-Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth.     

Alcohol increases mortality in murine head injury

Head injury is a major factor in the mortality of traumatized patients, accounting for about 50 percent of the resulting fatalities. Alcohol intoxication is frequently (25 to 50 percent) associated with head injuries. This study was undertaken to investigate the effects of alcohol on head trauma in a standardized animal model. Swiss Webster mice (25 ± 2 g) were given intraperitoneally 0.2 mL of either saline or 50 percent ethanol in saline. Thirty minutes later, under light ether anesthesia, severe concussion was produced by dropping a 39.5-g lead weight from a height of 30 cm. The trauma was centered on the midskull by channeling the weight through a vertical tube, 1.2 cm in diameter. Animals were observed daily for eight days. Among the controls, 12 of 12 mice, (100 percent) survived for four days and 8 of 12 (67 percent) survived eight days. In the alcohol recipients, there were 10 of 21 survivors (48 percent) at four days and only one survivor (5 percent) at eight days. This study clearly demonstrates that alcohol increases the lethality of standardized head trauma in mice. The mechanism by which alcohol modifies the effects of craniocerebral trauma remains to be elucidated.     

Lack of MK2 inhibits myofibroblast formation and exacerbates pulmonary fibrosis

Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2-/-) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2-/- mouse lungs, few alpha-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced alpha-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2-/- MEF. On the other hand expressing constitutively active MK2 in MK2-/- MEF significantly increased alpha-SMA expression. MK2-/-MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-beta. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.     

Type I interferon signaling exacerbates Chlamydia muridarum genital infection in a murine model

Type I interferons (IFNs) induced during in vitro chlamydial infection exert bactericidal and immunomodulatory functions. To determine the precise role of type I IFNs during in vivo chlamydial genital infection, we examined the course and outcome of Chlamydia muridarum genital infection in mice genetically deficient in the receptor for type I IFNs (IFNAR^−/− mice). A significant reduction in chlamydial shedding and duration of lower genital tract infection was observed in IFNAR^−/− mice in comparison to the level of chlamydial shedding and duration of infection in wild-type (WT) mice. Furthermore, IFNAR^−/− mice developed less chronic oviduct pathology in comparison to that in WT mice. Compared to the WT, IFNAR^−/− mice had a greater number of chlamydial-specific T cells in their iliac lymph nodes 21 days postinfection. IFNAR^−/− mice also exhibited earlier and enhanced CD4 T-cell recruitment to the cervical tissues, which was associated with increased expression of CXCL9 in the genital secretions of IFNAR^−/− mice, but not with expression of CXCL10, which was reduced in the genital secretions of IFNAR^−/− mice. These data suggest that type I IFNs exacerbate C. muridarum genital infection through an inhibition of the chlamydial-specific CD4 T-cell response.     

IL-6 deficiency exacerbates skin inflammation in a murine model of irritant dermatitis

Contact dermatitis is the second most reported occupational injury associated with workers compensation. Inflammatory cytokines are closely involved with the development of dermatitis, and their modulation could exacerbate skin damage, thus contributing to increased irritancy. IL-6 is a pro-inflammatory cytokine paradoxically associated with both skin healing and inflammation. To determine what role this pleiotropic cytokine plays in chemically-induced irritant dermatitis, IL-6 deficient (KO), IL-6 over-expressing transgenic (TgIL6), and corresponding wild-type (WT) mice were exposed to acetone or the irritants JP-8 jet fuel or benzalkonium chloride (BKC) daily for 7 days. Histological analysis of exposed skin was performed, as was tissue mRNA and protein expression patterns of inflammatory cytokines via QPCR and multiplex ELISA. The results indicated that, following JP-8 exposure, IL-6KO mice had greatly increased skin IL-1β, TNFα, CCL2, CCL3, and CXCL1 mRNA and corresponding product protein expression when compared to that of samples from WT counterparts and acetone-exposed control mice. BKC treatment induced the expression of all cytokines examined as compared to acetone, with CCL2 significantly higher in skin from IL-6KO mice. Histological analysis showed that IL-6KO mice displayed significantly more inflammatory cell infiltration as compared to WT and TgIL6 mice in response to jet fuel. Analysis of mRNA for the M2 macrophage marker CD206 indicated a 4-fold decrease in skin of IL-6KO mice treated with either irritant as compared to WT. Taken together, these observations suggest that IL-6 acts in an anti-inflammatory manner during irritant dermatitis, and these effects are dependent on the chemical nature of the irritant.     

Bone tuberculosis in a patient with pulmonary tuberculosis

A case of bone tuberculosis in 83 years old farmer was described. He was hospitalized because of pain, oedema and purulent fistula in carpal regio. He hadn't neither systemic symptoms neither from respiratory tract. Chest X-ray and X-ray of carpal bones revealed tuberculous lesions. In the pus from cutaneous fistula AFB were found in culture. Antituberculous treatment was successful.     

Transgenic tomato expressing interleukin-12 has a therapeutic effect in a murine model of progressive pulmonary tuberculosis

Host control of mycobacterial infection, in both human and mouse models, has been shown to be associated with the production of interferon (IFN)-gamma by CD4(+) T cells. Interleukin (IL)-12 is known to be a crucial cytokine in the differentiation of IFN-gamma-producing T helper 1 (Th1) cells. To determine whether continuous administration of IL-12 expressed in transgenic tomato (TT-IL-12) has therapeutic efficacy in a murine model of pulmonary tuberculosis, BALB/c mice were infected with either Mycobacterium tuberculosis H37Rv strain or a multi-drug-resistant clinical isolate (MDR) and treated with a daily oral dose of TT-IL12 crude fruit extracts. For the early H37Rv infection, TT-IL-12 administration was started 1 day before infection and continued for 60 days. In the H37Rv or MDR late infection, treatment was started 60 days after infection and continued for another 60 days. In both phases of infection, TT-IL-12 administration resulted in a reduction of bacterial loads and tissue damage compared with wild-type tomato (non-TT). The Th1 response was increased and the Th2 response was reduced. In the late infection, a long-term treatment with TT-IL-12 was necessary. We demonstrate that TT-IL-12 increases resistance to infection and reduces lung tissue damage during early and late drug-sensitive and drug-resistant mycobacterial infection.     

Experimental murine invasive pulmonary aspergillosis

A new model of invasive pulmonary aspergillosis (IPA) was developed in immunosuppressed mice. Intranasal route of inoculation was used to deliver predictable numbers of Aspergillus flavus conidia. The LD50 was determined to be 2.7 X 10(2) viable conidia, and a combination of quantitative culture and determination of chitin content was shown to best measure the progression of pulmonary disease. The evolution of IPA in these mice conformed with what has been reported in human cases of aspergillosis; both histopathology of the pulmonary lesions and dissemination pattern resembled their human counterparts. The authors hope to use this model to study virulence mechanisms of Aspergillus and novel therapeutic methods.