Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5 fl/fl LysM-Cre + mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5 fl/fl LysM-Cre + mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.utophagy is a fundamental cell biological process (1) with impact on aging, development, cancer, neurodegeneration, myodegeneration, metabolic disorders (2), idiopathic inflammatory diseases, and infection and immunity (3). Much of the physiological effects of autophagy are the result of degradative activities of autophagy (1), although biogenesis and secretory roles (4-6) of autophagy are beginning to be recognized (7). The execution of autophagy depends on factors collectively termed "Atg proteins," such as Atg5 (1) and Beclin 1 (Atg6) (8), whereas regulation of autophagy responds to various inputs via mammalian target of rapamycin (mTOR), including the presence of microbes (9), the TAB2/3-TAK1-IKK signaling axis (10), and processes downstream of pattern-recognition receptors and immune cytokine activation (3,(11)(12)(13).In the context of its immunological functions, autophagy acts in four principal ways (14). (i) Autophagy cooperates with conventional pattern-recognition receptors (PRRs), such as Toll-like receptors, RIG-I-like receptors (RLRs), and NOD-like receptors, and acts as both a regulator (11,12,15,16) and an effector of PRR signaling (17-19). (ii) Autophagy affects the presentation of cytosolic antigens in the context of MHC II molecules (20) in T-cell development, differentiation, polarization, and homeostasis (21,22). (iii) Most recently, autophagy has been shown to contribute to both the negative (6,7,(23)(24)(25) and positive (6, 7) regulation of unconventional secretion of the leaderless cytosolic proteins known as "alarmins," such as IL-1β and HMGB1. (iv) Autophagy can capture and eliminate intracellular microbes, including Mycobacterium tuberculosis (17, 26-29), which was one of the first two bacterial species (26, 30) to be recognized as targets for autophagic removal. This activity recently has been shown to depend on the recognition and capture of microbes by adaptors that represent a specialized subset of PRRs termed "sequestosome-like receptors" (SLRs) (31).M. tuberculosis is one of the first microbes recognized as being subject to elimination by immunological autophagy by murine and human...

Bacillus anthracis, the agent of anthrax, produces a poly-Dglutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD 50 and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.

Bioweapons are most often designed for delivery to the lung, although this route is not the usual portal of entry for many of the pathogens in the natural environment. Vaccines and therapeutics that are efficacious for natural routes of infection may not be effective against the pulmonary route. Pulmonary models are needed to investigate the importance of specific bacterial genes in virulence, to identify components of the host immune system that are important in providing innate and acquired protection, and for testing diagnostic and therapeutic strategies. This report describes the characteristics of host and Bacillus anthracis interactions in a murine pulmonary-infection model. The infective dose varied depending on the route and method of inoculation. The germination process in the lung began within 1 h of inoculation into the lung, although growth within the lung was limited. B. anthracis was found in the lung-associated lymph nodes ϳ5 h after infection. Minimal pneumonitis was associated with the lung infection, but significant systemic pathology was noted after dissemination. Infected mice typically succumbed to infection ϳ3 to 4 days after inoculation. The 50% lethal doses differed among inbred strains of mice, but within a given mouse strain, neither the age nor the sex of the mice influenced susceptibility to B. anthracis.

Phase I human clinical studies involving therapeutics for emerging and biodefense pathogens with low incidence, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), requires at a minimum preclinical evaluation of efficacy in two well-characterized and robust animal models. Thus, a ferret SARS-CoV model was evaluated over a period of 58 days following extensive optimization and characterization of the model in order to validate clinical, histopathological, virological and immunological endpoints. Ferrets that were infected intranasally with 10(3) TCID50 SARS-CoV showed higher body temperature (2-6 d.p.i.), sneezing (5-10 d.p.i.), lesions (5-7 d.p.i.) and decreased WBC/lymphocytes (2-5 d.p.i.). SARS-CoV was detected up to 7 d.p.i. in various tissues and excreta, while neutralizing antibody titers rose at 7 d.p.i. and peaked at 14 d.p.i. At 29 d.p.i., one group was challenged with 10(3) TCID50 SARS-CoV, and an anamnestic response in neutralizing antibodies was evident with no detectable virus. This study supports the validity of the ferret model for use in evaluating efficacy of potential therapeutics to treat SARS.

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Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD 50 ) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD 50 s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.