Comparative study of IgA VH3 gene usage in healthy TST− and TST+ population exposed to tuberculosis: deep sequencing analysis

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST⁺) and uninfected TST⁻) with M. tuberculosis. From a total of 22 V genes analysed, the TST⁻ population preferred the V_H3-23 and Vκ1 genes. The V_H3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST⁻ population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST⁺ population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST⁻ population. The antibodyome difference between both populations suggests a preference for antibodies with V_H3-23, D3-3, J_H4 gene usage by the TST⁻ population that could be associated with resistance to infection with M. tuberculosis.     

B Cells Modulate Systemic Responses to Pneumocystis murina Lung Infection and Protect On-Demand Hematopoiesis via T Cell-Independent Innate Mechanisms when Type I Interferon Signaling Is Absent

HIV infection results in a complex immunodeficiency due to loss of CD4⁺ T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag^−/−) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR^−/−) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag^−/− mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag^−/− mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4⁺ T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

Mycobacterium bovis BCG-Specific Th17 Cells Confer Partial Protection against Mycobacterium tuberculosis Infection in the Absence of Gamma Interferon

Protective immunity against tuberculosis (TB) requires the integrated response of a network of lymphocytes. Both gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-secreting CD4⁺ T cells have been identified in subjects with latent TB infection and during experimental Mycobacterium tuberculosis infection, but the contribution of Th17 cells to protective immunity is unclear. To examine their protective effects in vivo, we transferred mycobacterium-specific IL-17- and IFN-γ-secreting CD4⁺ T cells isolated from M. tuberculosis BCG-immunized IL-12p40^−/− and IFN-γ^−/− or wild-type mice, respectively, into M. tuberculosis-infected IL-12p40^−/− or RAG^−/− mice. In the absence of IL-12 and IL-23, neither IL-17-secreting (Th17) nor IFN-γ-secreting (Th1) BCG-specific T cells expanded or provided protection against M. tuberculosis. In RAG^−/− recipients with an intact IL-12/IL-23 axis, both Th17 and Th1 cells were activated and induced significant protection against M. tuberculosis. The reduction in the bacterial load following transfer of IFN-γ^−/− Th17 cells was associated with significant prolongation of survival compared to recipients of naïve IFN-γ^−/− T cells. This effect was at the cost of an increased inflammatory infiltrate characterized by an excess of neutrophils. Therefore, Th17 cells can provide IFN-γ-independent protection against M. tuberculosis, and this effect may contribute to the early control of M. tuberculosis infection.Protective immunity against Mycobacterium tuberculosis requires the orchestration and integration of innate and adaptive immune responses to generate a robust and long-lived memory T-cell response (17). Understanding the interactions of different types of lymphocytes in response to M. tuberculosis infection or the M. tuberculosis BCG vaccine is important for developing improved vaccines against M. tuberculosis and controlling the tuberculosis (TB) epidemic (29). M. tuberculosis infection stimulates dendritic cells (DCs) to secrete the cytokines interleukin 12 (IL-12) and IL-23, which promote the activation and clonal expansion of antigen-specific T cells in the draining lymph nodes (LN) of the lungs (4, 12). IL-12 and IL-23 are heterodimeric cytokines that have a shared p40 subunit, along with unique p35 and p19 subunits, respectively (18). IL-12 is required for the development of the gamma interferon (IFN-γ) T-cell response, which is essential for resistance to M. tuberculosis in both humans (7) and mice (4). On the other hand, mice lacking IL-23 can control M. tuberculosis infection (12). Although IL-23 is not essential for protection against M. tuberculosis, IL-23 may contribute to protection in the intact host, as plasmid IL-23 is as effective as plasmid IL-12 as an adjuvant to increase the protective response induced by a DNA vaccine (27).IL-23 has a major role in the expansion of IL-17-producing CD4⁺ T cells (9, 19). The initial activation of Th17 cells is dependent on IL-6 and TGF-β; however, in models of autoimmune disease, the expansion and proinflammatory effects of Th17 cells require IL-23 (1, 15, 25). Th17 cells secrete multiple cytokines, which induce chemokine production and the recruitment of neutrophils to mucosal sites of bacterial infection (28) and play an essential role in the control of oropharyngeal Candida albicans infection (3). Th17 cells also participate in the early inflammatory response to mycobacterial infection; however, IL-17 production in the lungs is downregulated as IFN-γ T cells emerge (5). The protective potential of Th17 T cells during the early phase of M. tuberculosis infection is unknown.Recently, we demonstrated that in the absence of the IL-12/IFN-γ axis, BCG-immunized IL-12p40^−/− mice developed a robust Th17 T-cell response. This was associated with a significant reduction in the bacterial load following infection with M. tuberculosis (26), suggesting that BCG-specific Th17 cells alone may provide partial protection in the absence of IFN-γ. To investigate this further, we expanded BCG-specific Th17 cells in vitro and examined their protective effects following adoptive transfer into M. tuberculosis-infected immunodeficient mice.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate of Salmonella typhimurium in Mice

The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv⁺ or Spv⁻ S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv⁺ versus Spv⁻ S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv⁺ and Spv⁻ salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv⁺ and Spv⁻ S. typhimurium strains. InvA⁻ Spv⁺ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA⁻ Spv⁻ salmonellae were only slightly attenuated relative to InvA⁺ Spv⁻ salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv⁻ S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.     

Hypercholesterolemia impairs immunity to tuberculosis

We demonstrate that apolipoprotein E -deficient (ApoE^−/−) mice are highly susceptible to tuberculosis and that their susceptibility depends on the severity of hypercholesterolemia. Wild-type (WT) mice and ApoE^−/− mice fed a low-cholesterol (LC) or high-cholesterol (HC) diet were infected with ~50 CFU Mycobacterium tuberculosis Erdman by aerosol. ApoE^−/− LC mice were modestly more susceptible to tuberculosis than WT LC mice. In contrast, ApoE^−/− HC mice were extremely susceptible, as evidenced by 100% mortality after 4 weeks with tuberculosis. The lung pathology of ApoE^−/− HC mice was remarkable for giant abscess-like lesions, massive infiltration by granulocytes, elevated inflammatory cytokine production, and a mean bacterial load ~2 log units higher than that of WT HC mice. Compared to WT HC mice, the gamma interferon response of splenocytes restimulated ex vivo with M. tuberculosis culture filtrate protein was delayed in ApoE^−/− HC mice, and they failed to control M. tuberculosis growth in the lung. OT-II cells adoptively transferred into uninfected ApoE^−/− HC mice had a weak proliferative response to their antigen, indicating impaired priming of the adaptive immune response. Our studies show that ApoE^−/− deficiency is associated with delayed expression of adaptive immunity to tuberculosis caused by defective priming of the adaptive immune response and that elevated serum cholesterol is responsible for this effect.     

Heme Catabolism by Heme Oxygenase-1 Confers Host Resistance to Mycobacterium Infection

Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (Mϕ) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1^−/−) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1^+/+) controls. Furthermore, Hmox1^−/− mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1^−/− versus Hmox1^+/+ SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected Mϕ, an effect mimicked by exogenous heme administration to M. avium-infected wild-type Mϕ in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in Mϕ, contributing critically to host resistance to Mycobacterium infection.     

Impaired Innate Immunity in Mice Deficient in Interleukin-1 Receptor-Associated Kinase 4 Leads to Defective Type 1 T Cell Responses,B Cell Expansion,and Enhanced Susceptibility to Infection with Toxoplasma gondii

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a member of the IRAK family and has an important role in inducing the production of inflammatory mediators. This kinase is downstream of MyD88, an adaptor protein essential for Toll-like receptor (TLR) function. We investigated the role of this kinase in IRAK4-deficient mice orally infected with the cystogenic ME49 strain of Toxoplasma gondii. IRAK4^−/− mice displayed higher morbidity, tissue parasitism, and accelerated mortality than the control mice. The lymphoid follicles and germinal centers from infected IRAK4^−/− mice were significantly smaller. We consistently found that IRAK4^−/− mice showed a defect in splenic B cell activation and expansion as well as diminished production of gamma interferon (IFN-γ) by T lymphocytes. The myeloid compartment was also affected. Both the frequency and ability of dendritic cells (DCs) and monocytes/macrophages to produce IL-12 were significantly decreased, and resistance to infection with Toxoplasma was rescued by treating IRAK4^−/− mice with recombinant IL-12 (rIL-12). Additionally, we report the association of IRAK4 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital toxoplasmosis in infected individuals (rs1461567 and rs4251513, P < 0.023 and P < 0.045, respectively). Thus, signaling via IRAK4 is essential for the activation of innate immune cells, development of parasite-specific acquired immunity, and host resistance to infection with T. gondii.     

Gamma Interferon Production by Cytotoxic T Lymphocytes Is Required for Resolution of Chlamydia trachomatis Infection

In this study, we used mice in which the gene for gamma interferon (IFN-γ) has been disrupted (IFN-γ^−/− mice) to study the role of this cytokine in the resolution of Chlamydia trachomatis infection. We show that IFN-γ^−/− mice are impaired in the ability to clear infection with C. trachomatis compared to IFN-γ^+/+ control mice. Activated CD8⁺ cytotoxic T lymphocytes (CTL) secrete IFN-γ in response to intracellular infection, and we have shown previously that a Chlamydia-specific CTL line can reduce C. trachomatis infection when adoptively transferred into infected mice. In the present study, we found that when these IFN-γ^+/+ CTL lines are transferred into Chlamydia-infected IFN-γ^−/− mice, the transferred CTL cannot overcome the immune defect seen in the IFN-γ^−/− mice. We also show that Chlamydia-specific CTL can be cultured from IFN-γ-deficient mice infected with C. trachomatis; however, the adoptive transfer of IFN-γ^−/− CTL into infected IFN-γ^+/+ mice does not reduce the level of infection. These results suggest that IFN-γ production by CTL is not sufficient to overcome the defect that IFN-γ^−/− mice have in the resolution of Chlamydia infection, yet IFN-γ production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-γ^+/+ mice.     

Interleukin-1 (IL-1) Signaling in Intestinal Stromal Cells Controls KC/CXCL1 Secretion,Which Correlates with Recruitment of IL-22-Secreting Neutrophils at Early Stages of Citrobacter rodentium Infection

Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli in humans and Citrobacter rodentium in mice, raise serious public health concerns. Here we demonstrate that interleukin-1 receptor (IL-1R) signaling is indispensable for protection against C. rodentium infection in mice. Four days after infection with C. rodentium, there were significantly fewer neutrophils (CD11b⁺ Ly6C⁺ Ly6G⁺) in the colons of IL-1R^−/− mice than in wild-type mice. Levels of mRNA and protein of KC/CXCL1 were also significantly reduced in colon homogenates of infected IL-1R^−/− mice relative to wild-type mice. Of note, infiltrated CD11b⁺ Ly6C⁺ Ly6G⁺ neutrophils were the main source of IL-22 secretion after C. rodentium infection. Interestingly, intestinal stromal cells isolated from IL-1R^−/− mice secreted lower levels of KC/CXCL1 than stromal cells from wild-type mice during C. rodentium infection. Similar effects were found when mouse intestinal stromal cells and human nasal polyp stromal cells were treated with IL-1R antagonists (i.e., anakinra) in vitro. These results suggest that IL-1 signaling plays a pivotal role in activating mucosal stromal cells to secrete KC/CXCL1, which is essential for infiltration of IL-22-secreting neutrophils upon bacterial infection.     

Type-I IFN Signaling Suppresses an Excessive IFN-γ Response and Thus Prevents Lung Damage and Chronic Inflammation During Pneumocystis (PC) Clearance in CD4 T Cell-Competent Mice

Immune-reconstitution after highly active antiretroviral therapy (HAART) is often incomplete, and some HIV-infected individuals fail to regenerate type-I interferon (IFN)–producing pDCs. We recently demonstrated that during Pneumocystis (PC) infection in CD4 T cell–competent mice the absence of type-I IFN signaling results in chronic pulmonary inflammation and fibrosis despite clearance. Because the mechanisms involved are poorly understood, we further characterized the role of type-I IFN signaling in immune responses to PC. We show that type-I IFN signaling around day 7 postinfection is critical to the outcome of inflammation. Microarray analysis of pulmonary CD11c⁺ cells revealed that at day 7 post infection, wild-type cells up-regulated type-I IFN–responsive genes as well as SOCS1, which is a critical negative-regulator of type-I IFN and IFN-γ signaling. This was associated with an eosinophilic lung inflammation, PC clearance, and complete restitution. However, pulmonary CD11c⁺ cells from IFNAR^−/− mice demonstrated increased tumor necrosis factor (TNF)-α production and lacked SOCS1-induction at day 7. This was followed by a transient lymphocytic and IFN-γ response before switching to a chronic eosinophilic inflammation of the lung. Early neutralization of TNF-α did not prevent chronic inflammation in IFNAR^−/− mice, but treatment with an anti–IFN-γ antibody did. We propose that during PC lung infection type-I IFNs induce SOCS1-associated regulatory mechanisms, which prevent excessive IFN-γ–mediated responses that cause chronic lung damage. Therefore, partial immune-reconstitution in AIDS, attributable to reduced type-I IFN actions, might disrupt regulatory aspects of inflammation, causing unexplained chronic pulmonary complications as seen in some patients during HAART.The advent of highly active antiretroviral therapy (HAART) has greatly improved immune functions in HIV-infected individuals and has led to a rapid reduction of opportunistic infections such as Pneumocystis pneumonia.^1,2 However, there is increasing recognition of other, noninfectious chronic pulmonary sequelae such as chronic obstructive lung diseases, asthma, pulmonary hypertension, and pulmonary neoplasms.³ The causes of these diseases are likely multifactorial, including immune-mediated mechanisms possibly because of lack of regulation.Immune reconstitution after HAART can be incomplete, as HIV infection not only causes the loss of CD4 ⁺ T cells, but results in a decrease of dendritic cell (DC) subpopulations such as type-I interferon (IFN)–producing plasmacytoid DC (pDC).^4,5 In some patients reconstitution of CD4 T cells may be satisfactory, but they may not adequately reconstitute DCs and specifically type-I IFN–producing pDC numbers.^6,7 The resulting lack of type I IFN production likely affects effector functions of cells of both innate and acquired immunity.⁸ How this may in addition influence immune regulatory aspects is not clear.Dendritic cells (DCs) and macrophages are important initiators and coordinators of both the innate and acquired immune responses. They can release large amounts of tumor necrosis factor (TNF)-α and/or type-I IFNs and responds to it.^9,10,11 In addition, cross regulatory mechanisms during inflammation are simultaneously initiated in these cells to prevent the unnecessary expansion of immune activation and tissue damage. Cytokine signaling also induces the expression of “suppressor of cytokine signaling molecules” (SOCS). These molecules function as negative regulators of inflammation by interfering with cytokine-initiated JAK-STAT–signaling pathways.^12,13 SOCS1 expression, for example, is initiated by and negatively regulates both type-I IFN and IFN-γ, and it has a specific and nonredundant role in the negative regulation of IFN-γ.We recently demonstrated in a mouse model of PC lung infection in CD4-competent mice that absence of IFNAR (type-I IFN receptor) results in a delayed but exacerbated TH2-mediated immune response, followed by lung fibrosis by day 35 post infection despite pathogen clearance when compared with wild-type mice in which the inflammation results in complete restitution.¹⁴ Here we further characterized the role of type-I IFN–signaling in regulating the immune response to PC lung infection in CD4-competent mice. We found that activation events near day 7 postinfection are critical in determining the outcome of inflammation. At this time CD11c⁺ alveolar macrophages and/or pulmonary CD11c⁺ DCs were the predominant inflammatory cells present in the lung. Microarray analysis of pulmonary CD11c⁺ cells at day 7 post-PC infections showed a transient type-I IFN–mediated gene expression signature in cells derived from wild-type mice, but not from IFNAR^−/− mice. This was associated with a concomitant upregulation of SOCS1 in pulmonary CD11c⁺ cells from wild-type mice which preceded a transient eosinophilic immune response at day 14 postinfection. In contrast, CD11c⁺ cells from IFNAR^−/− mice demonstrated a unique up-regulation of TNF-α at day 7 and also lacked SOCS1 expression at day 7. This was followed by a transient lymphocytic and IFN-γ–dominated inflammation by day 14 post infection before switching to an exacerbated and persisting eosinophilic inflammation. Neutralization of TNF-α during the course of infection did not prevent the chronic inflammation in IFNAR^−/− mice. However, treatment with neutralizing anti–IFN-γ antibody resulted in complete resolution of inflammation.The lack of a timely induction of negative regulators such as SOCS1 by type I IFN signaling in pulmonary CD11c⁺cells may be responsible for a transient but excessive and damaging IFN-γ response in IFNAR^−/− mice after PC infection. Partial immune reconstitution during HAART caused by reduced type-I IFN activity may also influence regulatory aspects of inflammation, which could promote chronic IFN-γ–mediated inflammatory sequellae.     

Proapoptotic Bcl-2 family member Bim promotes persistent infection and limits protective immunity

Following the peak of the T-cell response, most of the activated effector T cells die by apoptosis driven by the proapoptotic Bcl-2 family member Bim (Bcl-2-interacting mediator of death). Whether the absence of Bim-mediated T-cell apoptosis can affect protective immunity remains unclear. Here, we used a mouse model of Leishmania major infection, in which parasite persistence and protective immunity are controlled by an equilibrium reached between parasite-specific gamma interferon (IFN-γ)-producing effector T cells and interleukin-10 (IL-10)-producing CD4⁺ CD25⁺ T regulatory cells. To further understand the role of Bim-mediated apoptosis in persistent infection and protective immunity, we infected Bim^−/− mice with L. major. We found that the initial parasite growth and lesion development were similar in Bim^−/− and wild-type mice after primary L. major infection. However, at later times after infection, Bim^−/− mice had significantly increased L. major-specific CD4⁺ T-cell responses and were resistant to persistent infection. Interestingly, despite their resistance to primary L. major infection, Bim^−/− mice displayed significantly enhanced protection against challenge with L. major. Increased resistance to challenge in Bim^−/− mice was associated with a significant increase in the number of L. major-specific IFN-γ-producing CD4⁺ T cells and a lack of IL-10 production at the challenge site. Taken together, these data suggest that Bim limits protective immunity and that the absence of Bim allows the host to bypass antigen persistence for maintenance of immunity against reinfection.     

Gamma interferon-independent effects of interleukin-12 on immunity to Salmonella enterica serovar Typhimurium

Interleukin-12 (IL-12) and IL-18 are both central to the induction of gamma interferon (IFN-γ), and various roles for IL-12 and IL-18 in control of intracellular microbial infections have been demonstrated. We used IL-12p40^−/− and IL-18^−/− mice to further investigate the role of IL-12 and IL-18 in control of Salmonella enterica serovar Typhimurium. While C57BL/6 and IL-18^−/− mice were able to resolve attenuated S. enterica serovar Typhimurium infections, the IL-12p40^−/− mice succumbed to a high bacterial burden after 60 days. Using ovalbumin (OVA)-specific T-cell receptor transgenic T cells (OT-II cells), we demonstrated that following oral infection with recombinant S. enterica serovar Typhimurium expressing OVA, the OT-II cells proliferated in the mesenteric lymph nodes of C57BL/6 and IL-18^−/− mice but not in IL-12p40^−/− mice. In addition, we demonstrated by flow cytometry that equivalent or increased numbers of T cells produced IFN-γ in IL-12p40^−/− mice compared with the numbers of T cells that produced IFN-γ in C57BL/6 and IL-18^−/− mice. Finally, we demonstrated that removal of macrophages from S. enterica serovar Typhimurium-infected C57BL/6 and IL-12p40^−/− mice did not affect the bacterial load, suggesting that impaired control of S. enterica serovar Typhimurium infection in the absence of IL-12p40 is not due to reduced macrophage bactericidal activities, while IL-18^−/− mice did rely on the presence of macrophages for control of the infection. Our results suggest that IL-12p40, but not IL-18, is critical to resolution of infections with attenuated S. enterica serovar Typhimurium and that especially the effects of IL-12p40 on proliferative responses of CD4⁺ T cells, but not the ability of these cells to produce IFN-γ, are important in the resolution of infection by this intracellular bacterial pathogen.     

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.     

Bone Marrow Mesenchymal Stem Cells Provide an Antibiotic-Protective Niche for Persistent Viable Mycobacterium tuberculosis that Survive Antibiotic Treatment

During tuberculosis (TB), some Mycobacterium tuberculosis bacilli persist in the presence of an active immunity and antibiotics that are used to treat the disease. Herein, by using the Cornell model of TB persistence, we further explored our recent finding that suggested that M. tuberculosis can escape therapy by residing in the bone marrow (BM) mesenchymal stem cells. We initially showed that M. tuberculosis rapidly disseminates to the mouse BM after aerosol exposure and maintained a stable burden for at least 220 days. In contrast, in the lungs, the M. tuberculosis burden peaked at 28 days and subsequently declined approximately 10-fold. More important, treatment of the mice with the antibiotics rifampicin and isoniazid, as expected, resulted in effective clearance of M. tuberculosis from the lungs and spleen. In contrast, M. tuberculosis persisted, albeit at low numbers, in the BM of antibiotic-treated mice. Moreover, most viable M. tuberculosis was recovered from the bone marrow CD271⁺CD45⁻-enriched cell fraction, and only few viable bacteria could be isolated from the CD271⁻CD45⁺ cell fraction. These results clearly show that BM mesenchymal stem cells provide an antibiotic-protective niche for M. tuberculosis and suggest that unraveling the mechanisms underlying this phenomenon will enhance our understanding of M. tuberculosis persistence in treated TB patients.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an intracellular pathogen known to infect primarily macrophages and dendritic cells.^1–3 However, the viability of M. tuberculosis in these intracellular niches is poor,⁴ and no evidence exists indicating that these cells can maintain live nonreplicating M. tuberculosis for long periods of time. Therefore, it is unlikely that these cell populations can harbor viable M. tuberculosis during the chronic phase of the disease, which lasts for months or years, as well as during the latent TB infection, which can last for decades.We have recently shown that M. tuberculosis can reside in bone marrow (BM) within the CD271⁺CD45⁻ mesenchymal stem cells (BM-MSCs) of individuals treated for pulmonary TB and in mice experimentally infected with M. tuberculosis.⁵ MSCs can provide an ideal protective niche for M. tuberculosis because these cells have several properties that may promote the pathogen''s long-term persistence and survival: i) MSCs are present in TB granulomas of infected mouse and human lung tissue⁶; ii) stem cells possess the capacity for self-renewal⁷; iii) stem cells express drug efflux pumps, such as ABCG2, that could contribute to drug evasion by M. tuberculosis⁵; iv) stem cells have low production of reactive oxygen species,⁸ which may favor the viability of nonreplicating M. tuberculosis; v) although MSCs have the capacity of self-renewal, they are relatively quiescent,⁹ and reside in the immune-privileged niche of the BM^10,11; and vi) MSCs do not normally express major histocompatibility complex (MHC) class II on their cell surface and their MHC class I molecules are not functionally active (ie, these molecules do not trigger effector functions of cytotoxic T lymphocytes).¹² Therefore, it is logical that BM-MSCs constitute a host cell capable of supporting long-term persistence of viable nonreplicating M. tuberculosis. However, many fundamental questions regarding the survival of virulent M. tuberculosis in BM-MSCs remain unanswered. Herein, we confirmed in vivo in the mouse model of TB that virulent M. tuberculosis disseminates rapidly to the BM within 2 weeks after infection with aerosolized organisms and preferentially resides within BM-MSCs. In addition, and more important, we show that antibiotics appeared to efficiently clear the infectious process within the lungs and spleens but fail to do so in the BM.     

Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal?/? Mice

The liver is a major organ for lipid synthesis and metabolism. Deficiency of lysosomal acid lipase (LAL; official name Lipa, encoded by Lipa) in mice (lal^−/−) results in enlarged liver size due to neutral lipid storage in hepatocytes and Kupffer cells. To test the functional role of LAL in hepatocyte, hepatocyte-specific expression of human LAL (hLAL) in lal^−/− mice was established by cross-breeding of liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− knockout (KO) (LAP-Tg/KO) triple mice. Hepatocyte-specific expression of hLAL in LAP-Tg/KO triple mice reduced the liver size to the normal level by decreasing lipid storage in both hepatocytes and Kupffer cells. hLAL expression reduced tumor-promoting myeloid-derived suppressive cells in the liver of lal^−/− mice. As a result, B16 melanoma metastasis to the liver was almost completely blocked. Expression and secretion of multiple tumor-promoting cytokines or chemokines in the liver were also significantly reduced. Because hLAL is a secretory protein, lal^−/− phenotypes in other compartments (eg, blood, spleen, and lung) also ameliorated, including systemic reduction of myeloid-derived suppressive cells, an increase in CD4⁺ and CD8⁺ T and B lymphocytes, and reduced B16 melanoma metastasis in the lung. These results support a concept that LAL in hepatocytes is a critical metabolic enzyme in controlling neutral lipid metabolism, liver homeostasis, immune response, and tumor metastasis.Lysosomal acid lipase (LAL) (official name LIPA, encoded by LIPA) hydrolyzes cholesteryl esters (CEs) and triglycerides (TGs) in lysosomes. Mutation in LIPA results in Wolman disease (WD) as early infantile onset and CE storage disease (CESD) as late onset.^1–3 Infants with WD have massive accumulations of CEs and TGs in the lysosomes of hepatocytes and Kupffer cells, as well as in macrophages throughout the viscera, which lead to liver failure, severe hepatosplenomegaly, steatorrhea, pulmonary fibrosis, and adrenal calcification.² Patients with CESD have the major symptom of hepatomegaly with increased hepatic levels of CEs, which reveals microvesicular steatosis leading to fibrosis and cirrhosis in the liver and increases atherosclerosis and premature demise.^4–6An LAL knockout (KO) mouse model (lal^−/−) resembles human CESD, and its biochemical and histopathologic phenotypes mimic human WD.^7,8 The lal^−/− mice are normal appearing at birth but develop liver enlargement by 1.5 months and have a grossly enlarged abdomen with hepatosplenomegaly and lymph node enlargement.^7,8 One interesting character of lal^−/− mice is a systemic expansion (including the liver) of CD11b⁺Ly6G⁺ cells, which are similar to myeloid-derived suppressive cells (MDSCs) in tumorigenesis.^9–11 MDSCs influence the tissue microenvironment and contribute to local pathogenesis.^7–13 In humans, increased CD14⁺CD16⁺ and CD14⁺CD33⁺ cells (human subsets of MDSCs) have been linked to heterozygote carriers of LAL mutations.¹⁴ Hepatocellular carcinoma has been linked to chronic inflammation with elevated MDSC counts.^15–17To better understand the physiologic role of LAL in hepatocytes and the link to clinical application, hepatocyte-specific expression of human LAL (hLAL) in lal^−/− mice was achieved by crossbreeding a liver-activated promoter (LAP)–driven tTA transgene (LAP-tTA) and a (tetO)₇-CMV-hLAL transgene (Tet off system) into lal^−/− mice (LAP-Tg/KO mice). Histologic and tissue lipid analyses revealed a correction of lipid storage in the liver, spleen, and small intestine in doxycycline-untreated LAP-Tg/KO mice (hLAL induction turns on) compared with doxycycline-treated LAP-Tg/KO mice (hLAL induction turns off). Flow cytometry analyses revealed reduced tumor-promoting MDSCs in the liver of LAP-Tg/KO mice. Furthermore, hLAL overexpression in hepatocytes greatly reduced metastasis of B16 melanoma into the liver. These pathogenic phenotypes were associated with a decrease of inflammatory cytokine and chemokine levels. Together, these results indicate that hepatic LAL plays an important role in lipid metabolism, cytokine production, MDSCs influx into organs, and tumor metastasis in the liver.     

Liver Xeno-Repopulation with Human Hepatocytes in Fah?/?Rag2?/? Mice after Pharmacological Immunosuppression

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah^−/− Rag2^−/−Il2rg^−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah^−/−Rag2^−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah^−/−Rag2^−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.Human hepatocytes xeno-engrafted into the liver of immunodeficient mice could be used as a model to study human hepatitis virus infection in vivo as well as the efficacy of potential vaccines.^1,2,3,4,5,6 Engrafted human hepatocytes can be serially transplanted from primary mice into secondary mice without losing hepatic function.⁷ Mouse recipients of human liver cells must have two capabilities: robust liver repopulation and immune tolerance for human hepatocytes. Liver xeno-repopulation from human hepatocytes was first reported in uPA/Rag2^−/− mice¹ and uPA/SCID mice.^2,3,8 The levels of liver xeno-repopulation varies in several reports, ranging from 10% to as high as 90%.^1,8 Humanized livers in uPA/SCID mice are susceptible to hepatitis B virus (HBV)^1,2 and HCV^3,4 infection. However, uPA mice have several disadvantages: i) neonatal death during colony breeding; ii) transplantation of hepatocytes into newborn mice (within the second week of life) is technically difficult due to a bleeding disorder in the mice; iii) there is uncontrollable selection for donor cells; iv) there is autoreversion of endogenous hepatocytes; and v) kidney damage is induced by the human complement system.^1,2,8,9Recently, robust liver xeno-repopulation from human hepatocytes was found in Fah^−/−Rag2^−/−Il2rg^−/− mice, cross-bred from Fah^−/− mice and Rag2^−/−Il2rg^−/− mice.⁷ Fah^−/−Rag2^−/−Il2rg^−/− mice have advantages over previous immunodeficient uPA models.⁷ First, Rag2^−/−Il2rg^−/− mice lack B, T, and NK cells, rendering more complete immunodeficiency compared with either Rag2^−/− or SCID mice.¹⁰ Second, liver injury in Fah^−/− mice is controllable by switching on and off 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3 cyclohexanedione (NTBC) administration.¹¹ NTBC inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah^−/− mice in a healthy state. When the NTBC is removed, a powerful selection for fumaryl acetoacetate hydrolase (Fah) expressing cells is induced in the liver.¹² However, maintenance of Fah^−/−Rag2^−/−Il2rg^−/− mice during colony breeding, animal growing, and cell transplantation surgery are with high mortality in our experiments. The genotyping of animal offspring is overly elaborate. These concerns have not been discussed in previous publications^7,13 and may present a barrier to larger scale research projects.In comparison, Fah^−/−Rag2^−/− mice were much more tolerant of breeding and surgery procedures. However, Fah^−/−Rag2^−/− mice were thought to have no capacity for liver xeno-repopulation, because their NK cells are intact.⁷ We hypothesized that treatment of Fah^−/− Rag2^−/− mice with anti-asialo GM1 could result in complete depletion of NK cells as seen in Fah^−/−Rag2^−/− Il2rg^−/− mice.^14,15 We further tested the combined treatments of both anti-asialo GM1 and the immunosuppressor tacrolimus (FK506) to Fah^−/−Rag2^−/− mice.^16,17 The results indicated that the combined treatments enabled Fah^−/−Rag2^−/− recipients to have a high level of liver xeno-repopulation by human hepatocytes as seen in Fah^−/−Rag2^−/−Il2rg^−/− mice. Our results revealed a new and easily controlled mouse model with humanized liver. Using the same treatments, liver xeno-repopulation with human fetal liver progenitor cells was also achieved in Fah^−/−Rag2^−/− mice. Finally, for the first time, we were able to prove that human HBV actively replicated in the humanized Fah^−/−Rag2^−/− mice and that viral proteins were released in the serum of humanized Fah^−/−Rag2^−/− mice, which showed no significant difference with previous reports of human HBV infection in humanized uPA/SCID mice.^1,2,5     

CD14 Influences Host Immune Responses and Alternative Activation of Macrophages during Schistosoma mansoni Infection

Antigen-presenting cell (APC) plasticity is critical for controlling inflammation in metabolic diseases and infections. The roles that pattern recognition receptors (PRRs) play in regulating APC phenotypes are just now being defined. We evaluated the expression of PRRs on APCs in mice infected with the helminth parasite Schistosoma mansoni and observed an upregulation of CD14 expression on macrophages. Schistosome-infected Cd14^−/− mice showed significantly increased alternative activation of (M2) macrophages in the livers compared to infected wild-type (wt) mice. In addition, splenocytes from infected Cd14^−/− mice exhibited increased production of CD4⁺-specific interleukin-4 (IL-4), IL-5, and IL-13 and CD4⁺Foxp3⁺IL-10⁺ regulatory T cells compared to cells from infected wt mice. S. mansoni-infected Cd14^−/− mice also presented with smaller liver egg granulomas associated with increased collagen deposition compared to granulomas in infected wt mice. The highest expression of CD14 was found on liver macrophages in infected mice. To determine if the Cd14^−/− phenotype was in part due to increased M2 macrophages, we adoptively transferred wt macrophages into Cd14^−/− mice and normalized the M2 and CD4⁺ Th cell balance close to that observed in infected wt mice. Finally, we demonstrated that CD14 regulates STAT6 activation, as Cd14^−/− mice had increased STAT6 activation in vivo, suggesting that lack of CD14 impacts the IL-4Rα-STAT6 pathway, altering macrophage polarization during parasite infection. Collectively, these data identify a previously unrecognized role for CD14 in regulating macrophage plasticity and CD4⁺ T cell biasing during helminth infection.     

Interleukin-1 Receptor but Not Toll-Like Receptor 2 Is Essential for MyD88-Dependent Th17 Immunity to Coccidioides Infection

Interleukin-17A (IL-17A)-producing CD4⁺ T helper (Th17) cells have been shown to be essential for defense against pulmonary infection with Coccidioides species. However, we have just begun to identify the required pattern recognition receptors and understand the signal pathways that lead to Th17 cell activation after fungal infection. We previously reported that Card9^−/− mice vaccinated with formalin-killed spherules failed to acquire resistance to Coccidioides infection. Here, we report that both MyD88^−/− and Card9^−/− mice immunized with a live, attenuated vaccine also fail to acquire protective immunity to this respiratory disease. Like Card9^−/− mice, vaccinated MyD88^−/− mice revealed a significant reduction in numbers of both Th17 and Th1 cells in their lungs after Coccidioides infection. Both Toll-like receptor 2 (TLR2) and IL-1 receptor type 1 (IL-1r1) upstream of MyD88 have been implicated in Th17 cell differentiation. Surprisingly, vaccinated TLR2^−/− and wild-type (WT) mice showed similar outcomes after pulmonary infection with Coccidioides, while vaccinated IL-1r1^−/− mice revealed a significant reduction in the number of Th17 cells in their infected lungs compared to WT mice. Thus, activation of both IL-1r1/MyD88- and Card9-mediated Th17 immunity is essential for protection against Coccidioides infection. Our data also reveal that the numbers of Th17 cells were reduced in IL-1r1^−/− mice to a lesser extent than in MyD88^−/− mice, raising the possibility that other TLRs are involved in MyD88-dependent Th17 immunity to coccidioidomycosis. An antimicrobial action of Th17 cells is to promote early recruitment of neutrophils to infection sites. Our data revealed that neutrophils are required for vaccine immunity to this respiratory disease.