CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Assessing vaccine-mediated protection in an ultra-low dose Mycobacterium tuberculosis murine model.

Despite widespread immunization with Bacille-Calmette-Guérin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing.

Antibiotic Treatment during Gestation Enhances Susceptibility to Mycobacterium tuberculosis in Offspring.

Whether antibiotic treatment during gestation impacts T cell immunity to vaccination in offspring is unexplored. Dams treated with polymyxin B (PMB) during gestation (Mg) displayed altered microbial communities prior to delivery compared to control dams (Mc). Differences in microbiota were also evident in pups born to polymyxin B-treated dams (Pg) compared to control pups (Pc). When pups were immunized with Bacille Calmette-Guerin (BCG), we observed no difference in TB10.4-specific T cells between Pc and Pg 4 weeks postimmunization. Significantly fewer splenic CD4 T cells from BCG-vaccinated Pg produced interleukin-2 (IL-2) upon stimulation, suggesting a possible functional deficiency. There was no difference in purified protein derivative (PPD)-specific IgG between Pc and Pg at this time point. However, when infected with Mycobacterium tuberculosis, Pg displayed significantly higher bacterial burden in the lung than Pc. Our results show that maternal PMB treatment during gestation may not impact splenic antigen-specific T cell responses following BCG vaccination but alters susceptibility to M. tuberculosis in offspring. IMPORTANCE The composition of the pioneer microbiota that colonize the infant gut are determined by the mother. Polymyxin B-induced changes in the maternal microbiota during pregnancy impact the offspring gut microbiota but not vaccine-specific CD4 T cell response. However, when infected with Mycobacterium tuberculosis, offspring born to mothers with an altered gut microbiota are susceptible to infection compared to those born to mothers not exposed to antibiotics.

The Tuberculous Granuloma and Preexisting Immunity.

Pulmonary granulomas are widely considered the epicenters of the immune response to Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Recent animal studies have revealed factors that either promote or restrict TB immunity within granulomas. These models, however, typically ignore the impact of preexisting immunity on cellular organization and function, an important consideration because most TB probably occurs through reinfection of previously exposed individuals. Human postmortem research from the pre-antibiotic era showed that infections in Mtb-naïve individuals (primary TB) versus those with prior Mtb exposure (postprimary TB) have distinct pathologic features. We review recent animal findings in TB granuloma biology, which largely reflect primary TB. We also discuss our current understanding of postprimary TB lesions, about which much less is known. Many knowledge gaps remain, particularly regarding how preexisting immunity shapes granuloma structure and local immune responses at Mtb infection sites.

Ultra-low Dose Aerosol Infection of Mice with Mycobacterium tuberculosis More Closely Models Human Tuberculosis.

Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50-100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB.

Protective efficacy of an attenuated Mtb ΔLprG vaccine in mice.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Cutting Edge: Bacillus Calmette-Guérin-Induced T Cells Shape Mycobacterium tuberculosis Infection before Reducing the Bacterial Burden.

Growing evidence suggests the outcome of Mycobacterium tuberculosis infection is established rapidly after exposure, but how the current tuberculosis vaccine, bacillus Calmette-Guérin (BCG), impacts early immunity is poorly understood. In this study, we found that murine BCG immunization promotes a dramatic shift in infected cell types. Although alveolar macrophages are the major infected cell for the first 2 weeks in unimmunized animals, BCG promotes the accelerated recruitment and infection of lung-infiltrating phagocytes. Interestingly, this shift is dependent on CD4 T cells, yet does not require intrinsic recognition of Ag presented by infected alveolar macrophages. M. tuberculosis-specific T cells are first activated in lung regions devoid of infected cells, and these events precede vaccine-induced reduction of the bacterial burden, which occurs only after the colocalization of T cells and infected cells. Understanding how BCG alters early immune responses to M. tuberculosis provides new avenues to improve upon the immunity it confers.

Alveolar Macrophages Provide an Early Mycobacterium tuberculosis Niche and Initiate Dissemination.

Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, but the bacteria ultimately disseminate to the lung interstitium. Although various cell types, including alveolar macrophages (AM), neutrophils, and permissive monocytes, are known to be infected with Mtb, the initially infected cells as well as those that mediate dissemination from the alveoli to the lung interstitium are unknown. In this study, using a murine infection model, we reveal that early, productive Mtb infection occurs almost exclusively within airway-resident AM. Thereafter Mtb-infected, but not uninfected, AM localize to the lung interstitium through mechanisms requiring an intact Mtb ESX-1 secretion system. Relocalization of infected AM precedes Mtb uptake by recruited monocyte-derived macrophages and neutrophils. This dissemination process is driven by non-hematopoietic host MyD88/interleukin-1 receptor inflammasome signaling. Thus, interleukin-1-mediated crosstalk between Mtb-infected AM and non-hematopoietic cells promotes pulmonary Mtb infection by enabling infected cells to disseminate from the alveoli to the lung interstitium.

Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn's disease (CD). Consistent with this observation, laboratory mice with enterocyte-specific Sel1L deficiency (Sel1L(ΔIEC)) develop spontaneous enteritis and have increased susceptibility to Toxoplasma gondii-induced ileitis. This is associated with profound defects in Paneth cells and a disproportionate increase of Ruminococcus gnavus, a mucolytic bacterium with known association with CD. Surprisingly, whereas both ER stress sensor IRE1α and effector CHOP are activated in the small intestine of Sel1L(ΔIEC) mice, they are not solely responsible for ERAD deficiency-associated lesions seen in the small intestine. Thus our study points to a constitutive role of Sel1L-Hrd1 ERAD in epithelial cell biology and the pathogenesis of intestinal inflammation in CD.

Impact of Toxoplasma gondii on Dendritic Cell Subset Function in the Intestinal Mucosa.

The function of mucosal dendritic cell (DC) subsets in immunity and inflammation is not well understood. In this study, we define four DC subsets present within the lamina propria and mesenteric lymph node compartments based on expression of CD103 and CD11b. Using IL-12p40 YFP (Yet40) reporter mice, we show that CD103(+)CD11b(-) mucosal DCs are primary in vivo sources of IL-12p40; we also identified CD103(-)CD11b(-) mucosal DCs as a novel population producing this cytokine. Infection was preferentially found in CD11b(+) DCs that were negative for CD103. Lamina propria DCs containing parasites were negative for IL-12p40. Instead, production of the cytokine was strictly a property of noninfected cells. We also show that vitamin A metabolism, as measured by ALDH activity, was preferentially found in CD103(+)CD11b(+) DC and was strongly downregulated in all mucosal DC subsets during infection. Finally, overall apoptosis of lamina propria DC subsets was increased during infection. Combined, these results highlight the ability of intestinal Toxoplasma infection to alter mucosal DC activity at both the whole population level and at the level of individual subsets.

Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells.

Beta-catenin signaling has recently been tied to the emergence of tolerogenic dendritic cells (DCs). In this article, we demonstrate a novel role for beta-catenin in directing DC subset development through IFN regulatory factor 8 (IRF8) activation. We found that splenic DC precursors express beta-catenin, and DCs from mice with CD11c-specific constitutive beta-catenin activation upregulated IRF8 through targeting of the Irf8 promoter, leading to in vivo expansion of IRF8-dependent CD8a+, plasmacytoid, and CD103+ CD11b2 DCs. beta-catenin­stabilized CD8a+ DCs secreted elevated IL-12 upon in vitro microbial stimulation, and pharmacological beta-catenin inhibition blocked this response in wild-type cells. Upon infections with Toxoplasma gondii and vaccinia virus, mice with stabilized DC beta-catenin displayed abnormally high Th1 and CD8+ T lymphocyte responses, respectively. Collectively, these results reveal a novel and unexpected function for beta-catenin in programming DC differentiation toward subsets that orchestrate proinflammatory immunity to infection.

Influence of ethnicity and improved outcome of acute myeloid leukaemia: two decades of follow-up of Israeli patient cohort.

Acute myeloid leukaemia is a disease with unfavourable prognosis. The significance of various prognostic parameters is not fully understood. We studied 293 patients to examine the influence of ethnicity and molecular markers. The median survival for all patients was correlated with age, white blood cell count and karyotype, and marginally with FLT3 internal tandem duplication. Arab patients were younger than Jewish patients; however, their survival was poorer albeit being treated with the same protocols and having more favourable cytogenetics. Survival rates improved over time but only for patients undergoing allogeneic bone marrow transplantation (alloBMT). We conclude that in our young patient cohort, recent improvement in survival is attributed to alloBMT therapy and that ethnicity affected treatment outcome.

CXCR3-dependent CD4⁺ T cells are required to activate inflammatory monocytes for defense against intestinal infection.

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3⁻/⁻ mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3⁻/⁻ mice. Strikingly, adoptive transfer of wild-type but not Ifnγ⁻/⁻ CD4⁺ T lymphocytes into Cxcr3⁻/⁻ animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

Insights into inflammatory bowel disease using Toxoplasma gondii as an infectious trigger.

Oral infection of certain inbred mouse strains with the protozoan Toxoplasma gondii triggers inflammatory pathology resembling lesions seen during human inflammatory bowel disease, in particular Crohn's disease (CD). Damage triggered by the parasite is largely localized to the distal portion of the small intestine, and as such is one of only a few models for ileal inflammation. This is important because ileal involvement is a characteristic of CD in over two-thirds of patients. The disease induced by Toxoplasma is mediated by Th1 cells and the cytokines tumor necrosis factor-α and interferon-γ. Inflammation is dependent upon IL-23, also identified by genome-wide association studies as a risk factor in CD. Development of lesions is concomitant with emergence of E. coli that display enhanced adhesion to the intestinal epithelium and subepithelial translocation. Furthermore, depletion of gut flora renders mice resistant to Toxoplasma-triggered ileitis. Recent findings suggest complex CCR2-dependent interactions between lamina propria T cells and intraepithelial lymphocytes in fueling proinflammatory pathology in the intestine. The advantage of the Toxoplasma model is that disease develops rapidly (within 7-10 days of infection) and can be induced in immunodeficient mice by adoptive transfer of mucosal T cells from infected donors. We propose that Toxoplasma acts as a trigger setting into motion a series of events culminating in loss of tolerance in the intestine and emergence of pathogenic T cell effectors. The Toxoplasma trigger model is providing new leaps in our understanding of immunity in the intestine.

Absence of Rickettsia rickettsii and occurrence of other spotted fever group rickettsiae in ticks from Tennessee.

Rocky Mountain spotted fever (RMSF) is the most common tick-borne illness in Tennessee. Little is known about the occurrence of R. rickettsii, the causative agent, in ticks in Tennessee. To better understand the prevalence and distribution of rickettsial agents in ticks, we tested 1,265 Amblyomma, Dermacentor, and Ixodes adult and nymphal ticks. Additionally, we tested 231 Amblyomma americanum larvae. Ticks were collected from 49 counties from humans, wild animals, domestic canines, and flannel drags. Spotted fever group rickettsiae (SFGR) DNA was detected by polymerase chain reaction (PCR) in 32% of adult and nymphal ticks. A total minimum infection rate of 85.63 was found in larval pools tested. Three rickettsial species, Rickettsia montana, Rickettsia amblyommii, and Rickettsia cooleyi were identified by molecular analysis. Rickettsia rickettsii was not detected. This study suggests that some RMSF cases reported in Tennessee may be caused by cross-reactivity with other SFGR antigenically related to R. rickettsii.

Prevalence of Ehrlichia chaffeensis and Ehrlichia ewingii in ticks from Tennessee.

Human ehrlichiosis is the second most common tick-borne disease reported in Tennessee after Rocky Mountain spotted fever. Two closely related ehrlichiae, Ehrlichia chaffeensis and Ehrlichia ewingii, are both causative agents of human disease and are transmitted by Amblyomma americanum, the lone star tick. Prevalence rates and distribution patterns of these pathogens among ticks in Tennessee are currently unknown. To understand prevalence and exposure risk of Ehrlichia spp., we tested 616 ticks (309 Amblyomma americanum (L.), 277 Dermacentor variabilis (Say), 17 Ixodes texanus (Banks), 7 Ixodes cookei Packard, 4 Ixodes scapularis (Say), and 2 Amblyomma maculatum Koch) from 46 counties for E. chaffeensis and 324 ticks (238 A. americanum and 86 D. variabilis) from 29 counties for E. ewingii. Overall, E. chaffeensis was detected in 2.6% (8/309) of A. americanum and E. ewingii in 0.8% (2/238). Ehrlichia spp. DNA was not detected in any tick species other than A. americanum. Although sample sizes were low in many counties, all positive ticks were identified in the Interior Plateau and Southeastern Plains ecoregions which is where the majority of human ehrlichiosis cases are reported from Tennessee (e.g., 66.3% of the human cases in 2008 are from the Interior Plateau ecoregion). The data from this pathogen survey combined with frequent human case reports from certain areas indicate potential "hot spots" for ehrlichiosis infection. Targeted vector control interventions in these areas may help decrease human ehrlichiosis transmission.