Host innate and adaptive immunity shapes the gut microbiota biogeography.

The gut microbiota has a fundamental role in the development and the maturation of the host immune system. Both innate and adaptive immune cells have critical functions in microbial pathogen containment and clearance, but the regulation of the commensal microbiome ecosystem in the gastrointestinal tract by these major immune cell populations is incompletely defined. The role of specific innate and adaptive immune cell in the regulation of the microbiota in the intestinal tract biogeographically was investigated. Dendritic cells, macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells were depleted using monoclonal antibodies and clodronate liposomes, and the microbial communities were determined by 16S rRNA gene sequencing. With specific immune cell depletion, distinct microbiota changes were observed. In general, immune cell depleted mice had higher microbiota richness and evenness at all gut anatomical sites. At each gut segment, samples from immune cell-depleted animals clustered away from the isotype/liposome control mice. This was especially dramatic for the small intestinal microbiota. Specifically, Enterobacteriaceae, Bacteroides acidifaciens, and Mucispirillum schaedleri were highly enriched in the mucosa and lumen of the small intestine in immune cell-deficient animals. Further, the mucosal microbiota had higher microbiota evenness compared with luminal microbiota at all gut segments, and the UniFrac distance between B cell depleted and isotype control mice was the largest in the duodenum followed by the ileum and colon. Taken together, the data suggest that innate and adaptive immune cells specifically contribute to the regulation of the gut microbiota's biogeographical distribution along the gastrointestinal tract, and microbiota in the duodenum mucosa are more responsive to host immune changes compared with other anatomical sites.

Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function.

BACKGROUND: Chronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa-associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol-associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear. METHODS: To investigate the impact of alcohol on MAIT cells, mice were treated with binge-on-chronic alcohol for 10 days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol-induced MAIT cell changes: (1) Cecal microbiota from alcohol-fed (AF) mice were adoptive transferred into antibiotic-pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry. RESULTS: Binge-on-chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue-associated MAIT cells was increased in AF mice compared with pair-fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals. CONCLUSION: MAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol-associated dysbiosis. MAIT cell dysfunction may contribute to alcohol-induced innate and adaptive immunity and consequently end-organ pathophysiology.

Alcohol-associated intestinal dysbiosis impairs pulmonary host defense against Klebsiella pneumoniae.

Chronic alcohol consumption perturbs the normal intestinal microbial communities (dysbiosis). To investigate the relationship between alcohol-mediated dysbiosis and pulmonary host defense we developed a fecal adoptive transfer model, which allows us to investigate the impact of alcohol-induced gut dysbiosis on host immune response to an infectious challenge at a distal organ, independent of prevailing alcohol use. Male C57BL/6 mice were treated with a cocktail of antibiotics (ampicillin, gentamicin, neomycin, vancomycin, and metronidazole) via daily gavage for two weeks. A separate group of animals was fed a chronic alcohol (or isocaloric dextrose pair-fed controls) liquid diet for 10 days. Microbiota-depleted mice were recolonized with intestinal microbiota from alcohol-fed or pair-fed (control) animals. Following recolonization groups of mice were sacrificed prior to and 48 hrs. post respiratory infection with Klebsiella pneumoniae. Klebsiella lung burden, lung immunology and inflammation, as well as intestinal immunology, inflammation, and barrier damage were examined. Results showed that alcohol-associated susceptibility to K. pneumoniae is, in part, mediated by gut dysbiosis, as alcohol-naïve animals recolonized with a microbiota isolated from alcohol-fed mice had an increased respiratory burden of K. pneumoniae compared to mice recolonized with a control microbiota. The increased susceptibility in alcohol-dysbiosis recolonized animals was associated with an increase in pulmonary inflammatory cytokines, and a decrease in the number of CD4+ and CD8+ T-cells in the lung following Klebsiella infection but an increase in T-cell counts in the intestinal tract following Klebsiella infection, suggesting intestinal T-cell sequestration as a factor in impaired lung host defense. Mice recolonized with an alcohol-dysbiotic microbiota also had increased intestinal damage as measured by increased levels of serum intestinal fatty acid binding protein. Collectively, these results suggest that alterations in the intestinal immune response as a consequence of alcohol-induced dysbiosis contribute to increased host susceptibility to Klebsiella pneumonia.

Intestinal Microbial Products From Alcohol-Fed Mice Contribute to Intestinal Permeability and Peripheral Immune Activation.

BACKGROUND: Alcohol use causes significant disruption of intestinal microbial communities, yet exactly how these dysbiotic communities interact with the host is unclear. We sought to understand the role of microbial products associated with alcohol dysbiosis in mice on intestinal permeability and immune activation in an in vitro model system. METHODS: Microbiota samples from binge-on-chronic alcohol-fed and pair-fed male and female mice were cultured in Gifu Anaerobic Broth for 24 hours under anaerobic conditions. Live/whole organisms were removed, and microbial products were collected and added to human peripheral blood mononuclear cells (PBMCs) or polarized C2BBe1 intestinal epithelial monolayers. Following stimulation, transepithelial electrical resistance (TEER) was measured using a volt/ohm meter and immune activation of PBMC was assessed via flow cytometry. RESULTS: Microbial products from male and female alcohol-fed mice significantly decreased TEER (mean percentage change from baseline alcohol-fed 0.86 Ω/cm2 vs. pair-fed 1.10 Ω/cm2 ) compared to microbial products from control mice. Following ex vivo stimulation, immune activation of PBMC was assessed via flow cytometry. We found that microbial products from alcohol-fed mice significantly increased the percentage of CD38+ CD4+ (mean alcohol-fed 17.32% ± 0.683% standard deviation (SD) vs. mean pair-fed 14.2% ± 1.21% SD, p < 0.05) and CD8+ (mean alcohol-fed 20.28% ± 0.88% SD vs. mean pair-fed 12.58% ± 3.59% SD, p < 0.05) T cells. CONCLUSIONS: Collectively, these data suggest that microbial products contribute to immune activation and intestinal permeability associated with alcohol dysbiosis. Further, utilization of these ex vivo microbial product assays will allow us to rapidly assess the impact of microbial products on intestinal permeability and immune activation and to identify probiotic therapies to ameliorate these defects.

The respiratory tract microbial biogeography in alcohol use disorder.

Individuals with alcohol use disorders (AUDs) are at an increased risk of pneumonia and acute respiratory distress syndrome. Data of the lung microbiome in the setting of AUDs are lacking. The objective of this study was to determine the microbial biogeography of the upper and lower respiratory tract in individuals with AUDs compared with non-AUD subjects. Gargle, protected bronchial brush, and bronchoalveolar lavage specimens were collected during research bronchoscopies. Bacterial 16S gene sequencing and phylogenetic analysis was performed, and the alterations to the respiratory tract microbiota and changes in microbial biogeography were determined. The microbial structure of the upper and lower respiratory tract was significantly altered in subjects with AUDs compared with controls. Subjects with AUD have greater microbial diversity [ P < 0.0001, effect size = 16 ± 1.7 observed taxa] and changes in microbial species relative abundances. Furthermore, microbial communities in the upper and lower respiratory tract displayed greater similarity in subjects with AUDs. Alcohol use is associated with an altered composition of the respiratory tract microbiota. Subjects with AUDs demonstrate convergence of the microbial phylogeny and taxonomic communities between distinct biogeographical sites within the respiratory tract. These results support a mechanistic pathway potentially explaining the increased incidence of pneumonia and lung diseases in patients with AUDs.

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Analysis of the intestinal microbial community and inferred functional capacities during the host response to Pneumocystis pneumonia.

BACKGROUND: Pneumocystis pneumonia is a major cause of morbidity and mortality in patients infected with HIV/AIDS. In this study, we evaluated the intestinal microbial communities associated with the development of experimental Pneumocystis pneumonia, as there is growing evidence that the intestinal microbiota is critical for host defense against fungal pathogens. METHODS: C57BL/6 mice were infected with live Pneumocystis murina (P. murina) via intratracheal inoculation and sacrificed 7 and 14 days postinfection for microbiota analysis. In addition, we evaluated the intestinal microbiota from CD4+ T cell depleted mice infected with P. murina. RESULTS: We found that the diversity of the intestinal microbial community was significantly altered by respiratory infection with P. murina. Specifically, mice infected with P. murina had altered microbial populations, as judged by changes in diversity metrics and relative taxa abundances. We also found that CD4+ T cell depleted mice infected with P. murina exhibited significantly altered intestinal microbiota that was distinct from immunocompetent mice infected with P. murina, suggesting that loss of CD4+ T cells may also affects the intestinal microbiota in the setting of Pneumocystis pneumonia. Finally, we employed a predictive metagenomics approach to evaluate various microbial features. We found that Pneumocystis pneumonia significantly alters the intestinal microbiota's inferred functional potential for carbohydrate, energy, and xenobiotic metabolism, as well as signal transduction pathways. CONCLUSIONS: Our study provides insight into specific-microbial clades and inferred microbial functional pathways associated with Pneumocystis pneumonia. Our data also suggest a role for the gut-lung axis in host defense in the lung.

Human Immunodeficiency Virus Infection and Host Defense in the Lungs.

Immunosuppression associated with human immunodeficiency virus (HIV) infection impacts all components of host defense against pulmonary infection. Cells within the lung have altered immune function and are important reservoirs for HIV infection. The host immune response to infected lung cells further compromises responses to a secondary pathogenic insult. In the upper respiratory tract, mucociliary function is impaired and there are decreased levels of salivary immunoglobulin A. Host defenses in the lower respiratory tract are controlled by alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes. As HIV infection progresses, lung CD4 T cells are reduced in number causing a lack of activation signals from CD4 T cells and impaired defense by macrophages. CD8 T cells, on the other hand, are increased in number and cause lymphocytic alveolitis. Specific antibody responses by B-lymphocytes are decreased and opsonization of microorganisms is impaired. These observed defects in host defense of the respiratory tract explain the susceptibility of HIV-infected persons for oropharyngeal candidiasis, bacterial pneumonia, Pneumocystis pneumonia, and other opportunistic infections.

Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

Cross sectional analysis of the effect of alcohol on pulmonary function in a cohort of men and women living with HIV.

People living with HIV (PLWH) are at increased risk for noncommunicable diseases such as lung disease in part due to opportunistic infections including pneumonia. HIV infection is associated with increased prevalence of impaired lung function and abnormal gas exchange. Alcohol use disorder (AUD) is exceedingly common in PLWH and is associated with higher risk of pneumonia in PLWH. Alcohol use may lead to lung damage through several mechanisms. Data on the long-term effect of AUD on pulmonary function in PLWH are sparse and conflicting. To evaluate this relationship, we conducted a cross-sectional analysis of adult PLWH in care in Louisiana. We hypothesized that chronic alcohol use would be associated with subsequent pulmonary dysfunction in a dose-dependent fashion. All participants performed standardized spirometry on study entry. In total, 350 participants with acceptable spirometry were included in this analysis. Thirty-one percent of participants were female. Women reported less lifetime alcohol use and less smoking; however, they reported more chronic respiratory symptoms. In adjusted models, total lifetime alcohol use was not associated with spirometry measures of pulmonary function. HIV-related variables (CD4 count and viral load) were also not associated with measures of pulmonary function. We then conducted sex-stratified analyses to eliminate residual confounding of sex and similarly found no association of total lifetime alcohol use and pulmonary function. We found no association of AUDIT score or early life alcohol use and pulmonary function. In latent class factor analysis, current heavy alcohol use was associated with lower measures of pulmonary function as compared to former heavy alcohol use. In summary, in this cohort of New Orleanian men and women living with HIV with robust measures of alcohol use, though total lifetime alcohol use and early life alcohol use were not associated with pulmonary function, current heavy alcohol use was associated with impaired pulmonary function.

Thymopoietic and bone marrow response to murine Pneumocystis pneumonia.

CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.

Role of interleukin-23-dependent antifungal immune responses in dendritic cell-vaccinated mice.

CD40 ligand (CD40L) transduction of antigen-pulsed dendritic cells (DCs) can result in antigen-specific humoral immune responses even in CD4(+) T-cell-depleted settings. Here, we show that CD40L transduction of DCs results in the induction of interleukin-12p40 (IL-12p40), IL-12p70, and IL-23. Using DCs that were deficient in IL-12p40, IL-12p35, or IL-23p19, we show that these molecules are dispensable for primary IgG1 responses to Pneumocystis, but IgG2c was dependent on IL-12p40 and IL-23p19 but not IL-12p35. Antigen-specific recall responses in CD4-deficient mice were critically dependent on IL-12p40 and IL-23p19 expression in DCs and were not affected by the lack of IL-12p35. To confirm that this defect in recall was due to IL-23, transduction of IL-12p40(-/-) DCs with a recombinant adenovirus expressing functional IL-23 restored recall responses in DC-vaccinated CD4-deficient mice. These data show that DC-produced IL-23 is critical for vaccine-induced antigen-specific IgG2c and recall antibody responses in the setting of CD4(+) T-cell depletion.

Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae.

Interleukin (IL)-23 is a heterodimeric cytokine that shares the identical p40 subunit as IL-12 but exhibits a unique p19 subunit similar to IL-12 p35. IL-12/23 p40, interferon gamma (IFN-gamma), and IL-17 are critical for host defense against Klebsiella pneumoniae. In vitro, K. pneumoniae-pulsed dendritic cell culture supernatants elicit T cell IL-17 production in a IL-23-dependent manner. However, the importance of IL-23 during in vivo pulmonary challenge is unknown. We show that IL-12/23 p40-deficient mice are exquisitely sensitive to intrapulmonary K. pneumoniae inoculation and that IL-23 p19-/-, IL-17R-/-, and IL-12 p35-/- mice also show increased susceptibility to infection. p40-/- mice fail to generate pulmonary IFN-gamma, IL-17, or IL-17F responses to infection, whereas p35-/- mice show normal IL-17 and IL-17F induction but reduced IFN-gamma. Lung IL-17 and IL-17F production in p19-/- mice was dramatically reduced, and this strain showed substantial mortality from a sublethal dose of bacteria (10(3) CFU), despite normal IFN-gamma induction. Administration of IL-17 restored bacterial control in p19-/- mice and to a lesser degree in p40-/- mice, suggesting an additional host defense requirement for IFN-gamma in this strain. Together, these data demonstrate independent requirements for IL-12 and IL-23 in pulmonary host defense against K. pneumoniae, the former of which is required for IFN-gamma expression and the latter of which is required for IL-17 production.

Pulmonary immune cell trafficking promotes host defense against alcohol-associated Klebsiella pneumonia.

The intestinal microbiota generates many different metabolites which are critical for the regulation of host signaling pathways. In fact, a wide-range of diseases are associated with increased levels of local or systemic microbe-derived metabolites. In contrast, certain bacterial metabolites, such as tryptophan metabolites, are known to contribute to both local and systemic homeostasis. Chronic alcohol consumption is accompanied by alterations to intestinal microbial communities, and their functional capacities. However, little is known about the role of alcohol-associated dysbiosis on host defense against bacterial pneumonia. Our previous work using fecal transplantation demonstrated that alcohol-associated intestinal dysbiosis, independent of ethanol consumption, increased susceptibility to Klebsiella pneumonia. Here, we demonstrate that intestinal microbiota treatments mitigate the increased risk of alcohol-associated pneumonia. Treatment with the microbial metabolite indole or with probiotics reduced pulmonary and extrapulmonary bacterial burden, restored immune responses, and improved cellular trafficking required for host defense. Protective effects were, in part, mediated by aryl hydrocarbon receptors (AhR), as inhibition of AhR diminished the protective effects. Thus, alcohol appears to impair the production/processing of tryptophan catabolites resulting in immune dysregulation and impaired cellular trafficking. These data support microbiota therapeutics as novel strategies to mitigate the increased risk for alcohol-associated bacterial pneumonia.

Acute effect of inhaled iloprost on exercise dynamic hyperinflation in COPD patients: A randomized crossover study.

BACKGROUND AND OBJECTIVE: We tested whether the prostacyclin analog inhaled iloprost modulates dead space, dynamic hyperinflation (DH), and systemic inflammation/oxidative stress during maximal exercise in subjects with chronic obstructive pulmonary disease (COPD) who were not selected based on pulmonary hypertension (PH). METHODS: Twenty-four COPD patients with moderate-severe obstruction (age 59 ± 7 years, FEV1 53 ± 13% predicted) participated in a randomized, double-blind, placebo-controlled crossover trial. Each subject received a single nebulized dose of 5.0 µg iloprost or placebo on non-consecutive days followed by maximal cardiopulmonary exercise tests. The primary outcome was DH quantified by end-expiratory lung volume/total lung capacity ratio (EELV/TLC) at metabolic isotime. RESULTS: Inhaled iloprost was well-tolerated and reduced submaximal alveolar dead-space fraction but did not significantly reduce DH (0.70 ± 0.09 vs 0.69 ± 0.07 following placebo and iloprost, respectively, p = 0.38). Maximal exercise time (9.1 ± 2.3 vs 9.3 ± 2.2 min, p = 0.31) and peak oxygen uptake (17.4 ± 6.3 vs 17.9 ± 6.9 mL/kg/min, p = 0.30) were not significantly different following placebo versus iloprost. CONCLUSIONS: A single dose of inhaled iloprost was safe and reduced alveolar dead space fraction; however, it was not efficacious in modulating DH or improving exercise capacity in COPD patients who were not selected for the presence of PH.

Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor.

Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 beta-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage-mediated killing of P. carinii. The macrophage Dectin-1 beta-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti-Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis. We further show that Dectin-1 is required for P. carinii-induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

The lineage-c-Kit+Sca-1+ cell response to Escherichia coli bacteremia in Balb/c mice.

During bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for host defenses. Along with this enhancement of granulopoiesis, the bone marrow also increases its release of hematopoietic precursors. At the present time, little is known about the commitment of hematopoietic precursor cells, including hematopoietic stem cells and progenitors, in this response. To investigate the hematopoietic precursor cell response to bacterial infection, bacteremia was established in Balb/c mice by i.v. injection of Escherichia coli. Bacteremia caused a 10-fold increase in the number of lineage (lin)-c-kit+Sca-1+ cells in the bone marrow. This dramatic expansion of the lin-c-kit+Sca-1+ cell pool resulted from both increased mitosis of these cells and inversion from lin-c-kit+Sca-1- cell phenotype. Lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 were potent factors capable of mediating phenotypic inversion of lin-c-kit+Sca-1- cells. Cells in the expanded lin-c-kit+Sca-1+ cell pool contained more colony-forming unit-granulocyte/macrophage. Mobilization of lin-c-kit+Sca-1+ cells into the circulation was significantly enhanced following bacteremia. These results demonstrate that the lin-c-kit+Sca-1+ cell population in the bone marrow constitutes a key component of the host defense response to bacteremia. Functional modifications of these primitive hematopoietic precursors are critical for enhancing granulocyte production following bacterial infection.

Lymphocyte apoptosis in murine Pneumocystis pneumonia.

BACKGROUND: Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. METHODS: Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. RESULTS: In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. CONCLUSION: Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Alcohol consumption increases susceptibility to pneumococcal pneumonia in a humanized murine HIV model mediated by intestinal dysbiosis.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Interleukin-12 and host defense against murine Pneumocystis pneumonia.

Little is known about the role of the cytokine interleukin-12 (IL-12) in Pneumocystis pneumonia or its potential use as immunotherapy. We asked whether release of IL-12 is part of the normal host response to this infection and whether local treatment with IL-12 or gene transfer of IL-12 could accelerate clearance of infection. IL-12 was assayed by enzyme-linked immunosorbent assay in normal mice and in mice deficient in IL-12 after inoculation of Pneumocystis carinii. P. carinii-infected mice were treated with local instillation of IL-12 and gene transfer of the IL-12 gene. Inoculation of P. carinii into normal mice evoked a brisk release of IL-12 into lung tissue, and IL-12 P35-deficient mice showed delayed clearance of infection measured by PCR for P. carinii rRNA. In control mice, intranasal recombinant IL-12 accelerated clearance of infection, and this was associated with increased recruitment of inflammatory cells into lavage fluid and increased release of tumor necrosis factor alpha, IL-12, and gamma interferon. Similar results were observed in infected mice depleted of CD4+ lymphocytes by using in vivo transfer of the IL-12 gene in a replication-deficient adenoviral vector. IL-12 is part of the normal host response to infection with P. carinii. IL-12 therapy can enhance host resistance to infection in both normal mice and mice depleted of CD4+ T lymphocytes. A treatment effect of IL-12 is mediated through enhanced inflammatory cell recruitment into lung tissue and increased tissue concentrations of proinflammatory cytokines.