Tuberculosis Mycobacterium Immune Response to Diabetic Mice Display

Diabetic Mice Display a Delayed Adaptive Immune Response to Mycobacterium tuberculosis

This information is current as Therese Vallerskog, Gregory W. Martens and Hardy of September 26, 2021. Kornfeld J Immunol 2010; 184:6275-6282; Prepublished online 26 April 2010; doi: 10.4049/jimmunol.1000304

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Supplementary http://www.jimmunol.org/content/suppl/2010/04/23/jimmunol.100030 Material 4.DC1 http://www.jimmunol.org/ References This article cites 34 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/184/11/6275.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Diabetic Mice Display a Delayed Adaptive Immune Response to Mycobacterium tuberculosis

Therese Vallerskog, Gregory W. Martens, and Hardy Kornfeld

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. We previously reported that streptozotocin-induced DM impaired TB defense in mice, resulting in higher pulmonary bacterial burden, more extensive inflammation, and higher expression of several proinflammatory cytokines known to play a protective role in TB. In the current study, we tested the hypothesis that DM leads to delayed priming of adaptive immunity in the lung- draining lymph nodes (LNs) following low dose aerosol challenge with virulent Mycobacterium tuberculosis. We show that M. tuberculosis-specific IFN-g–producing T cells arise later in the LNs of diabetic mice than controls, with a proportionate delay in recruitment of these cells to the lung and stimulation of IFN-g–dependent responses. Dissemination of M. tuberculosis from lung to

LNs was also delayed in diabetic mice, although they showed no defect in dendritic cell trafﬁcking from lung to LNs after Downloaded from LPS stimulation. Lung leukocyte aggregates at the initial sites of M. tuberculosis infection developed later in diabetic than in nondiabetic mice, possibly related to reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early time points postinfection. We conclude that TB increased susceptibility in DM results from a delayed innate immune response to the presence of M. tuberculosis-infected alveolar macrophages. This in turn causes late delivery of Ag-bearing APC to the lung draining LNs and delayed priming of the adaptive immune response that is necessary to restrict M. tuberculosis replication. The

Journal of Immunology, 2010, 184: 6275–6282. http://www.jimmunol.org/

pproximately one third of the world’s population is in- 439 million by 2030 (3). Of particular concern is the rapid increase fected with Mycobacterium tuberculosis, the causative in DM in developing countries with a high burden of TB (4). A agent of tuberculosis (TB), but this infection remains Despite the public health threat posed by these convergent epi- latent throughout life in ∼90% of infected individuals. For those demics, the immunological basis for TB susceptibility in DM is not ∼10% of M. tuberculosis-infected persons who develop active well understood. Wepreviouslypublished the first report oflow-dose disease, the inability to mount or to maintain an immune response aerosol M. tuberculosis infection in mice with streptozotocin that enforces latent TB infection results from diverse genetic and (STZ)-induced DM (5). Mice with chronic ($12 wk) but not acute acquired risk factors. Among the latter, HIV/AIDS has received (,4 wk) DM had a higher plateau bacterial burden and more lung by guest on September 26, 2021 much attention due to the dramatic increase in relative risk asso- inflammation by 8 wk postinfection (p.i.) compared with eugly- ciated with CD4+ T cell deficiency and the observation that TB is cemic controls. The finding that acute STZ-induced hyperglycemia the leading cause of death in that population (1). It has been rec- and hypoinsulinemia have no major impact on TB defense sug- ognized for some time that diabetes mellitus (DM) increases the gested that impaired host defense in DM could, like many other risk for TB, but the magnitude of this effect has only recently been diabetic complications, result from biochemical effects of pro- appreciated. In a review of 13 observational studies addressing the longed hyperglycemia. Despite evidence of increased susceptibil- association of DM and TB disease, Jeon and Murray (2) determined ity, mice with chronic DM and infected with M. tuberculosis for .4 that the global population-attributable risk is comparable to that of wk exhibited no evident deficiency in leukocyte recruitment to the HIV/AIDS. Although the immune dysfunction associated with DM lungs or in the expression of key cytokines relevant to TB defense. is not as severe as that caused by HIV infection, DM is a more On the contrary, diabetic mice had significantly more pulmonary common disorder than HIV/AIDS. The worldwide prevalence of T cells, macrophages, and neutrophils than controls, whereas the DM in 2010 is estimated to be 285 million and is predicted to reach relative proportions of these different populations were comparable to the controls. Similarly, the diabetic mice had more IFN-g, IL-1b, and TNF-a protein in their lungs than control mice at 8 wk or longer Department of Medicine, University of Massachusetts Medical School, Worcester, after M. tuberculosis infection. Mice with chronic DM were ca- MA 01655 pable of arresting M. tuberculosis replication in the lung, but at .1 Received for publication January 28, 2010. Accepted for publication March 22, 2010. log higher plateau bacterial burden than controls. Although diabetic This work was supported in part by National Institutes of Health Grant HL081149 (to mice had expressed more IFN-g than controls at later time points, H.K.). Core resources supported by the Diabetes Endocrinology Research Center they had significantly lower IFN-g expression 2 wk p.i., suggesting Grant DK32520 were also used. that TB susceptibility might result at least in part from a delay in Address correspondence and reprint requests to Dr. Hardy Kornfeld, Department of initiating M. tuberculosis-specific adaptive immunity. Medicine, LRB-303, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: [email protected] In the current study, we sought to gain further insight to the basis The online version of this article contains supplemental material. of TB susceptibility in mice with chronic STZ-induced DM (STZc). Abbreviations used in this paper: Ctrl, control; DC, dendritic cell; DM, diabetes We used STZ for these experiments to produce uniform hypergly- mellitus; HbA1c, hemoglobin A1c; i.t., intratracheal; LN, lymph node; na, not avail- cemia without the potentially confounding effects of autoimmunity able; p.i., postinfection; PPD, purified protein derivative; STZ, streptozotocin; STZc, in NOD mice that might interfere with the interpretation of any mice with chronic STZ-induced DM; TB, tuberculosis. observed TB susceptibility and in view of the fact that hypergly- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 cemia drives most diabetic complications. We report in this study www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000304 6276 DELAYED TUBERCULOSIS IMMUNITY IN DIABETIC MICE that the earliest appearance of M. tuberculosis-specific IFN-g– the expression of CD3, CD4, CD8, F4/80, Gr-1, and CD19. No differences producing T cells in the thoracic lymph nodes (LNs) and subse- were observed between control and STZc mice in the total number of these quently in the lung occurred later in STZc mice than control mice. different leukocyte types prior to M. tuberculosis challenge or at days 7, 14, 21, and 28 p.i. (Supplemental Fig. 2A). This is consistent with our prior Delivery of M. tuberculosis bacilli to the pulmonary LNs was also observations (5). Leukocytes from lung-draining LNs were evaluated for delayed in STZc mice but we found no evidence for impaired the expression of CD11c, CD19, CD4, and CD8 at days 12, 15, 18, and 21 dendritic cell (DC) trafficking from the lung to the LNs in STZc p.i., with no proportionate differences observed between control and STZc mice challenged with intratracheal (i.t.) LPS, nor any difference in mice (Supplemental Fig. 2B). the expression of MHC class II or costimulatory molecules on mi- Ex vivo T cell restimulation grating DCs. Examination of lung histopathology from early time IFN-g secreted from lung leukocytes and thoracic LN lymphocytes was points after M. tuberculosis infection demonstrated a delay in re- detected using an IFN-g Ab ELISPOT pair (BD Biosciences, San Jose, CA), cruitment of myeloid cells to sites of initial macrophage infection in MultiScreen-IP ELISPOT plates (Millipore, Billerica, MA), streptavidin- the lungs of STZc mice. In a similar time frame, these mice also had alkaline phosphatase (Mabtech, Nacka Strand, Sweden), 5-bromo-4-chloro- reduced expression of CCL2 and CCL5, chemokines that stimulate 3-indolyl phosphate/nitro blue tetrazolium, BCIP/NBT (Sigma-Aldrich), and M. tuberculosis Ag85 peptide (Ag85A243–260 sequence Ac-QDAYN- migration of macrophages and DCs. These findings pinpoint a criti- AGGGHNGVFDFPSDG-amide; Twentyfirst Century Biochemicals, Marl- cal lesion in protective immunity resulting from chronic hypergly- boro, MA) for stimulation of T cells. The membranes of ELISPOT plates cemia, wherein resident alveolar macrophages initially infected with were treated with 15 ml 70% ethanol, followed by eight washes with water M. tuberculosis after aerosol challenge fail to recruit naive myeloid and then coated with detection Ab diluted 1:250 in PBS overnight at 4˚C. cells necessary to acquire Ag and convey it to the pulmonary LN. We The plates were washed with PBS and the membrane blocked with complete cell culture medium (RPMI 1640 with 10% heat inactivated FCS, 2 mM Downloaded from conclude that events preceding the priming of Ag-specific T cells are L-glutamine, 50 mM 2-ME, 100 U/ml penicillin, 100 mg/ml streptomycin responsible for the delay in mounting what is otherwise a robust sulfate) for minimum 2 h in room temperature after which the wells were adaptive immune response against M. tuberculosis in diabetic mice. prepared with complete medium 6 10 mΜ Ag85 peptide, followed by addition of cells in triplicate for each cell concentration and stimulation. Uncoated wells and wells without cells were used as negative controls and Materials and Methods cells stimulated with 2 mg/ml Con A (Sigma-Aldrich), as positive controls. Mice After 44 h incubation (37˚C, 5% CO2), cells were washed off the membrane

with PBS and biotinylated detection Ab (1:200 in PBS-T80) was added and http://www.jimmunol.org/ C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, incubated for 2 h at room temperature. The wells were washed with PBS- ME). Mice were housed in the Animal Medicine facility at University of T80 and PBS before incubation with streptavidin-alkaline phosphatase for Massachusetts Medical School where experiments were performed under 1 h at room temperature, followed by PBS wash and addition of BCIP/NBT protocols approved by the Institutional Animal Care and Use Committee substrate. IFN-g spots were developed for 7–12 min before a final wash with and Institutional Biosafety Committee. water. The membrane was dried overnight at room temperature and the ELISPOT plates were decontaminated at 65˚C for 2 h. Analysis was per- Induction of DM formed with an automated ELISPOT reader (ImmunoSpot, CTL, Shaker DM was established by i.p. injection of STZ (Sigma-Aldrich, St. Louis, MO) Heights, OH), using CTL ImmunoSpot Academic Software version 4.0. Results were calculated as spots per 105 CD4+ T cells, based on the fre- dissolvedinphosphatecitratebuffer(pH4.5).Micewere atleast8 wkoldwith + minimumweightof25gwhentreatedwithSTZ.Bloodglucosemeasurements quency of CD4 T cells in LNs (analyzed by flow cytometry) and mean by guest on September 26, 2021 were performed with a BD Logic glucometer (Becton Dickinson, Franklin number of spots from each triplicate. Lakes, NJ) 9 d after STZ injection and immediately prior to M. tuberculosis Cytokine and NO analysis infection. Mice were considered diabetic if their blood glucose was .200 mg/dl. Mice with ketoacidosis, tested by dip stick (LW Scientific, Lawren- Lungs were homogenized in PBS-T80 and an equal volume of tissue protein ceville, GA), were excluded from the study. Mice were maintained with DM extraction reagent (T-PER, Pierce, Rockford, IL) was added and the mixture for 12 wk before other experimental manipulations were started; a time when was vortexed, incubated (20 min, 4˚C), vortexed again, centrifuged (10 hemoglobin A1c (HbA1c) was elevated (Supplemental Fig. 1). HbA1c was min, 14,0003g), and the supernatant was filter sterilized. Lung lysates measured in whole blood by affinity chromatography (Helena GLYCO-Tek; were assayed for IFN-g by ELISA according to manufacturer’s protocol Helena Laboratories, Beaumont, TX). (R&D Systems, Minneapolis, MN). NO (quantified as the sum of nitrite and nitrate) was detected in lung lysates by NO Quantitation Kit (Active M. tuberculosis infection Motif, Carlsbad, CA) by manufacturer’s protocol. Briefly, lung lysates were added to a 96-well plate, followed by addition of cofactors and nitrate Aliquots of frozen M. tuberculosis Erdman stock in PBS plus 0.05% reductase. After 30 min incubation, Griess Reagents were added to each Tween-80 (PBS-T80) were thawed and sonicated for 5 min in a cup-horn sample and absorbance at 540 nm was read 20 min later. Chemokines in sonifier (Branson Ultrasonics, Danbury, CT). Mice were infected by lung lysates were screened with a Multi-Analyte ELISArray Kit (SABio- aerosol with settings titrated to deliver ∼100 CFU per mouse using a Glas- sciences, Frederick, MD) according to manufacturer’s protocol. Pooled Col Inhalation Exposure System (Terre Haute, IN). Two mice were sac- lung lysate from each experimental group was added to the ELISArray rificed 24 h p.i. to confirm the actual delivered dose in every experiment. plate and incubated for 2 h. The plate was washed and detection Ab was Bacterial load added and incubated for 1 h and washed again. Bound Ab was detected by streptavidin-HRP and, after addition of the HRP substrate, absorbance was Thoracic LNs were harvested and then homogenized in deionized water plus read at 570 nm using a Thermo-Multiskan Ascent ELISA reader with 0.05% Tween 80 using a pestle grinder system with pestles for 1.5 ml tubes Ascent Software v.2.6 (Thermo Labsystems OY, Vantaa, Finland). In- (Fisher Scientific, Pittsburgh, PA). The homogenate was plated in duplicate dividual lung homogenate samples were analyzed for CXCL9 (MIG) by on Middlebrook 7H11 agar (DIFCO; Beckton Dickinson, Sparks, MD) and ELISA (R&D Systems), whereas CCL5 (RANTES), CCL2 (MCP-1), cultured at 37˚C for 3 wk. Colonies were counted using a dissecting mi- CXCL12 (SDF-1), and CCL17 (TARC) in individual samples were ana- croscope to confirm colony morphology. lyzed by a commercial multiplex sandwich-ELISA system (SearchLight Protein Array Technology, Aushon Biosystems, Billerica, MA). Cell preparation DC migration from lungs to LNs Lungs were perfused through the heart with PBS, minced, and digested at 37˚C in collagenase IV (1 mg/ml) and DNase (25 mg/ml; both from Sigma- To induce activation and cell migration from lungs to draining LNs, 0.5 mg Aldrich). After 45 min incubation, the digested lungs were passed through LPS (Sigma-Aldrich) and 1 mM CellTrace FarRed DDAO-SE (Invitrogen, a 40-mm cell strainer, washed, and remaining RBC were lysed with Gey’s Carlsbad, CA) was given in PBS in a total volume of 50 ml by i.t. instillation. solution (Sigma-Aldrich). Thoracic LNs were minced and digested at 37˚C Thoracic LNs were harvested after 20 h and leukocytes extracted from the in collagenase IV (0.02 mg/ml) and DNase (0.5 mg/ml) for 40 min and tissue by enzymatic digestion as described previously. Migrating FarRed+ passed through a 70-mm cell strainer. Viable cells were counted using CD11c+ cells were detected by flow cytometry using the following mAbs: a hemocytometer with dead cells exclusion by trypan blue (Sigma-Aldrich) CD11b-Pacific Blue (clone M1/70), CD11c-PE (clone N418), MHC class II staining. Lung leukocyte populations were evaluated by flow cytometry for (I-A/I-E)-Alexa 700 (clone M5/114.15.2), CD40-PE Cy5 (clone 1C10), The Journal of Immunology 6277

CD86-PE Cy7 (clone GL-1), CD83-FITC (clone Michel-17), CD80-Biotin (clone 16-10A1), streptavidin-Alexa 350 (Invitrogen). All mAbs were purchased from eBioscience (San Diego, CA), except CD86, which was purchased from BioLegend (San Diego, CA). The cells were blocked with BD Fc-block Ab (BD Bioscience) and stained 20 min with Abs after wash with FACS stain/wash buffer (1% FBS, 0.05% sodium azide). Streptavidin- Alexa 350 was added to the samples after washing, incubated 15 min, and washed a final time. A minimum 100,000 events within the live gate based on forward light scatter and side light scatter was collected with BD LSR II analyzer using BD FACSDiva v.5.0.3 software and data were analyzed using FlowJo v.7.2.5 software (Tree Star, Ashland, OR). Isotype control Ab was purchased from BD Pharmingen (San Diego, CA) and eBioscience. Histopathology Lungs were inflated and fixed with 10% buffered formalin for 24 h and then processed for staining. Paraffin embedded tissue sections were stained with H&E for histopathology and with a 1:100 dilution of anti-purified protein derivative (PPD) polyclonal rabbit IgG (Abcam, Cambridge, MA) for detection of M. tuberculosis. Enzymatic Ag retrieval was performed prior to immunostaining and Ab was detected with polyclonal goat HRP- conjugated anti-rabbit IgG (Dako, Carpinteria,CA). Lung sections were stained with an irrelevant rabbit IgG (Abcam) for negative controls. All staining was performed by the Diabetes and Endocrinology Research Downloaded from Center histopathology core facility at University of Massachusetts Medical School. Stained lung sections were analyzed with a Nikon Eclipse E400 microscope (Nikon Instruments, Melville, NY) using Spot Advanced v.4.6 software (Diagnostic Instruments, Sterling Heights, MN). Percent total lung area involved with inflammation was calculated by dividing the cu- FIGURE 1. Detection of M. tuberculosis-specific IFN-g–producing mulative area of inflammation by the total lung surface area examined in T cells in STZc and control (Ctrl) mice. Ex vivo reactivity of pulmonary LN all sections for each lung studied as we previously described (5). http://www.jimmunol.org/ (A) and lung leukocytes (B)toM. tuberculosis Ag85B was measured by Statistical analysis ELISPOT. Bars illustrate the number of IFN-g spots per 105 CD4+ cells at M. tuberculosis Statistical analysis was performed using Graph Pad Prism v.5.02 (Graphpad the indicated times after aerosol infection of nondiabetic Software, La Jolla, CA) software. Normally distributed data were analyzed control mice (filled bars) and diabetic STZc mice (empty bars). Bars rep- with Student t test or with Welch’s unpaired t test if variances were different. resent mean 6 SD, n = 3–8. Lines within each bar correspond to the value One-tailed t tests were performed on samples early p.i. where difference was for unstimulated cells for each group and time point. pp , 0.05; ppp , 0.01. expected on the higher levels above zero. Nonparametric data were analyzed with the Mann-Whitney U test. Categorical data were tested with Fisher exact test. p values # 0.05 were considered statistically significant. adaptive immune response to M. tuberculosis in diabetic mice, we reasoned that late delivery of M. tuberculosis to the lung-draining by guest on September 26, 2021 Results LNs might be responsible. To test this prediction, thoracic LNs Kinetics of adaptive immunity to M. tuberculosis in LNs and lungs were harvested from control and STZc mice every 48 h between To elucidate the impact of DM on the induction of M. tuberculosis- days 5 through 11 after low-dose aerosol M. tuberculosis challenge specific cell-mediated immunity, we challenged STZc mice and and the entire LN homogenate from each mouse was plated for nondiabetic controls by low-dose aerosol infection with virulent M. colony counting. Cultures from all mice tested at day 5 p.i. were tuberculosis Erdman. Leukocytes were extracted from the lungs negative for CFU. On day 7 p.i., 6 of 10 LNs in nondiabetic controls and lung-draining LNs on successive days p.i. and restimulated ex were positive for bacterial growth, whereas 3 of 9 diabetic mice had vivo with an H-2b restricted M. tuberculosis Ag85 peptide for IFN-g positive LN cultures at that time (Fig. 3). By day 9 p.i., the fre- ELISPOT (Fig. 1). In control mice, IFN-g spot-forming cells were quency of mice with detectable CFU was similar between the first detected in pulmonary LNs on day 12 p.i. and in the lung by day groups, and by day 11 p.i. 100% of the mice in both groups had 15 p.i. By comparison, IFN-g spot-forming cells were not detected positive LN cultures. In addition to having fewer positive LN cul- in LNs of STZc mice until day 15 p.i. and in the lung on day 21 p.i. tures on day 7 p.i., the diabetic mice displayed a lower bacterial Thus, the priming of naive T cells in thoracic LNs after aerosol M. load at early time points of dissemination (Table I). On day 9 p.i., tuberculosis challenge is delayed by ∼3 d in diabetic mice, with the range of CFU among positive samples was 6–196 (median 43) a corresponding late appearance of these activated cells in the lung in the nondiabetic control mice and 1–85 (median 17) in STZc parenchyma. The late priming of IFN-g T cells and their delayed mice. These results suggested that late delivery of M. tuberculosis recruitment to the lungs was functionally significant, as evidenced Ag in the form of viable bacilli contributed to the delayed priming by reduced lung tissue levels of IFN-g protein and of NO in STZc of adaptive immunity in diabetic mice with TB. mice on day 15 p.i. (Fig. 2A), as well as lower levels of the IFN-g– DC migration and expression of costimulatory molecules in inducible chemokine CXCL9 (Fig. 2B). The late arrival of IFN-g– response to LPS producing T cells at the site of infection occurs during a period of logarithmic bacterial replication in the lung and accounts for the Although resident alveolar macrophages are the initial host cell .10-fold higher plateau bacterial load that we previously reported population infected after inhalation of M. tuberculosis, there is in STZc mice (5). strong evidence that DCs convey bacilli to the lung-draining LNs where priming of adaptive immunity takes place (8). Various re- Bacterial dissemination from lung to draining LNs ports have associated DC dysfunction with DM (9–11) leading us to The induction of adaptive immunity to M. tuberculosis after aerosol speculate that the delayed priming of adaptive immunity in diabetic challenge depends on bacterial dissemination from the initial site of mice with TB might result from impaired DC trafficking to the LNs, infection in the lungs to the draining LNs where naive Ag-specific possibly coupled with reduced expression of MHC class II and/or precursor T cells are primed (6, 7). Because we observed a delayed costimulatory molecules required to activate T cells. To evaluate 6278 DELAYED TUBERCULOSIS IMMUNITY IN DIABETIC MICE

Pulmonary histopathology After inhalation of M. tuberculosis, lesions form in the lungs at sites where bacilli invade and replicate inside resident alveolar macrophages. We surveyed H&E stained lung sections from STZc and control mice over a time course of 8–28 d after low-dose aerosol M. tuberculosis infection (Fig. 5A). Lung lesions developed sooner in the control mice as compared with STZc mice and these early lesions on average were larger in the controls. Lesions were identified in some control mice by day 12 p.i. and by day 15 p.i. nearly all had visible lesions, whereas only a few of the diabetic mice had detectable lesions by 15 d p.i. (Fig. 5B). By day 28 p.i., pulmonary inflammation in the STZc mice was comparable to the nondiabetic group. This is consistent with our previous study where pulmonary TB immunopathology in STZc mice was equivalent to controls 4 wk p.i., but substantially greater in the diabetic group at 8 and 16 wk p.i. (5). We next evaluated lung lesions in relation to foci of M. tuberculosis infection by immunohistochemical staining with anti-PPD Ab and H&E counterstaining. M. tuberculosis-infected macro- Downloaded from phages were detectable in control and STZc mice at day 15 p.i. The infected macrophages were typically surrounded by many newly recruited myeloid cells in the control mice, but the lung sections FIGURE 2. Levels of IFN-g and host defense factors induced by IFN-g from diabetic mice in many instances had heavily infected mac- in the lungs of control and diabetic mice. A, Lungs were harvested from rophages with few recruited leukocytes in their vicinity (Fig. 6). nondiabetic Ctrl mice (filled circles) and STZc mice (empty circles) 15 d This finding reflects a diabetes-related defect in the innate response http://www.jimmunol.org/ after low-dose aerosol M. tuberculosis infection. Lung lysates were pre- to initial M. tuberculosis infection of resident alveolar macro- pared for measurement of IFN-g by ELISA (left panel) and nitrate/nitrate phages. Diabetic mice ultimately developed leukocyte aggregates as a reflection of NO (right panel). B, Levels of the IFN-g–inducible at foci of positive anti-PPD staining that were similar in appearance chemokine CXCL9 were measured by ELISA in lung homogenates of to those of control mice (Supplemental Fig. 3). diabetic and nondiabetic mice 15 d p.i. pp , 0.05. Pulmonary chemokines The slow development of leukocyte aggregates around M. tubercu- DC migration, we labeled airway resident leukocytes by i.t. in- losis-infected macrophages in diabetic mice suggested that innate stillation of the fluorescent dye FarRed and activated DCs by co- signals required for the evolution of such lesions might be deficient. by guest on September 26, 2021 delivery of LPS. Leukocytes from lung-draining LNs were We performed a multiplex ELISArray to survey levels of 12 che- harvested 20 h later, and the recently emigrated DCs were identified mokines in pooled samples of lung homogenate from STZc and + + as FarRed CD11c cells. Contrary to our expectation, there was no control mice (4 mice per group) on days 12, 15, and 18 p.i. (Fig. 7A, + + difference in the proportion of FarRed CD11c cells from LNs of Supplemental Fig. 4). By this method, control mice exhibited higher STZc mice or controls (Fig. 4B), nor any difference in their relative levels of CXCL9/MIG than STZc mice, consistent with previous expression of MHC class II, CD40, CD80, or CD86 (Fig. 4C). ELISA result (Fig. 2B). The ELISArray also indicated possible dif- Although LPS stimulation does not precisely mimic the pathways ferences in the levels of several chemokines including CCL2/MCP- of DC activation and migration from the lungs in TB, these results 1, CCL5/RANTES, CXCL10/IP-10, CXCL12/SDF-1, and CCL17/ suggested that the delayed delivery of M. tuberculosis bacilli and TARC that were all higher in control than in STZc mice. We next immune priming observed in STZc mice were not attributable to analyzed the concentrations of individual chemokines (CCL2, defects in DC migration from the lung to the LNs or their cos- CCL5, CCL17, and CXCL12) in unpooled lung samples from day 15 timulatory function. p.i. using a multiplex ELISA system. By this method, levels of CCL2 and CCL5 were higher in control mice than STZc mice (Fig. 7B). This finding may be relevant to delayed recruitment of leukocytes to

Table I. Dissemination of M. tuberculosis to the lung-draining LNs

5 d p.i. 7 d p.i. 9 d p.i. 11 d p.i. % LNs positive for CFU Ctrl 0 60 89 100 STZc 0 33 89 100 Mean CFU Ctrl 0 1 43 1180 STZc 0 0 17 1520 Range CFU Ctrl na 1–6 6–196* 230–384 STZc na 2–4 1–85 281–2150 n Ctrl 11 10 9 9 STZc 9 9 9 5 FIGURE 3. Dissemination of M. tuberculosis from lung to LNs. Non- diabetic Ctrl mice (ﬁlled circles) and STZc mice (open circles) were in- Nondiabetic (Ctrl) mice and mice with $3 mo STZc were infected with ∼100 fected with ∼100 CFU of M. tuberculosis Erdman by aerosol. Lung- CFU M. tuberculosis Erdman by aerosol and then lung-associated LNs were harvested for plating and colony counting on the indicated days p.i. draining LNs were isolated on the indicated days, homogenized, and plated pp , 0.05 (one-tailed t test). for determination of CFU. pp , 0.05. na, not available. The Journal of Immunology 6279

myeloid cells surrounding M. tuberculosis-infected alveolar macrophages, were seen earlier in control mice than in STZc mice. Reduced expression of certain chemokines was also noted at time points preceding the widespread expression of adaptive immunity in the lung. Delivery of M. tuberculosis to the lung-draining LNs occurred later in STZc than control mice, with a corresponding delay in the detection of T cells primed to produce IFN-g in response to M. tuberculosis Ag in the LNs initially and subsequently in the lung. The physiological consequence of reduced pulmonary IFN-g was supported by the ﬁnding of reduced IFN-g–inducible factors including NO (the product of inducible NO synthase) and CXCL9 in the diabetic mice. These changes may not be the only defense functions impaired by chronic hyperglycemia but they could explain several features of TB disease in diabetic mice, including their capacity to induce stationary persistence of M. tuberculosis but at a much higher bacterial load than controls, with concomitantly increased chronic inﬂammation at later time points. Human TB is primarily a disease of the lung, which is the initial

site of infection in nearly all cases. Evading (and/or suppressing) Downloaded from immunity allows the bacillus to establish a foothold in the host before the expression of IFN-g and other defense factors forces a shift in bacterial gene expression and function resulting in stationary persistence (12). The lung appears to be uniquely vul- nerable to TB. High-dose i.v. M. tuberculosis challenge in mice

delivers logs more bacteria to the liver and spleen than the lung, http://www.jimmunol.org/ but after several weeks bacterial burden in the lung exceeds that in other organs and pulmonary inflammation becomes the main feature disease (13). Low-dose aerosol infection of mice is followed by an ∼3 wk period of logarithmic M. tuberculosis growth in the lung that is constrained only after a Th1-biased adaptive immune response is expressed (14, 15). The slow kinetics of adaptive immunity in TB differs from the response to many other respiratory infections. Chackerian et al. (6) reported slower M. tuberculosis dissemination from lung to tho- by guest on September 26, 2021 racic LNs in TB-susceptible C3H mice compared with the more resistant strain C57BL/6, and that dissemination preceded priming of adaptive immunity. Studies using fluorescent M. bovis BCG or fluorescent M. tuberculosis H37Rv (8, 16), or using adoptive transfer of fluorescently-labeled DCs and macrophages pulsed in vitro with irradiated M. tuberculosis H37Rv (17), established that DCs preferentially carry bacilli to the LNs. Work from several groups confirms that immunity to M. tuberculosis after aerosol FIGURE 4. DC migration from lung to LNs in response to LPS. Pul- challenge is primed in the lung-draining LNs (7, 17–19), although monary DCs were simultaneously labeled with FarRed and activated with priming can occur at other sites in mice lacking LNs (20) or lacking LPS by i.t. instillation. After 20 h, the lung-draining LNs were removed and leukocytes were analyzed by flow cytometry. A, Gating strategy to CCR7 (21). Our data presented in this paper suggest that DM ex- identify newly emigrated DC as FarRed+ CD11c+ cells. B, Proportion of acerbates the already slow pace of generating immunity to M. tu- FarRed+ CD11c+ cells in LNs of control and STZc mice 20 h after LPS berculosis. This effect of DM could shed light on key factors stimulation. C, Expression of CD40, CD80, CD86, and MHC class II on regulating the kinetics of TB immunity in the euglycemic host. a gated population of FarRed+ CD11c+ cells. The sluggish development of myeloid aggregates around M. tuberculosis-infected macrophages in the lungs of diabetic mice suggests deficiency of signals that alert the immune system to the foci of M. tuberculosis infection because these two chemokines presence of infecting bacilli. Macrophages infected by inhaled M. promote migration of DCs and monocyte/macrophages. tuberculosis use chemokines to recruit naive macrophages and DCs to the site of infection. Mice deficient in CCR2 exhibit delayed Discussion recruitment of macrophages and DCs to the lung after aerosol M. We previously reported that mice with chronic, but not acute, hy- tuberculosis challenge, along with delayed expression of IFN-g and perglycemia had increased TB susceptibility evidenced by higher inducible NO synthase (22, 23). Mice lacking the CCR2 ligand pulmonary bacterial burden and more widespread immunopathol- CCL2 also exhibited reduced pulmonary macrophage recruitment ogy (5). A robust Th1-biased cell mediated immune response de- and IFN-g expression in the aerosol TB model (24). In our study, veloped after 4 wk p.i. with many key factors for successful TB CCL2 levels in the lung on day 15 p.i. were lower in STZc than defense expressed in abundance. Experiments in the current study nondiabetic control mice. It is unlikely that deficient CCL2 ex- tested the hypothesis that TB susceptibility in STZc mice is due to pression alone fully accounts for the impaired TB defense of di- delayed initiation of adaptive immunity. The first lung lesions de- abetic mice, because their susceptibility appears to be greater than tectable by light microscopy after aerosol challenge, aggregates of that reported for CCR2 or CCL2 null mice. Along with reduced 6280 DELAYED TUBERCULOSIS IMMUNITY IN DIABETIC MICE Downloaded from

FIGURE 5. Pulmonary lesions at early time points after M. tuberculosis infection. Ctrl and STZc mice were challenged with ∼100 CFU M. tuberculosis Erdman by aerosol and the lungs were subsequently harvested, inflated, and fixed with formalin and then processed for H&E staining. A, Leukocyte 3 B aggregates (arrows) were easily identified in nondiabetic Ctrl mice but not in mice with chronic DM (STZc) by microscopy at 20 magnification. , Total http://www.jimmunol.org/ cross-sectional lung area from all tissues sections examined (3–6 mice per group at different time points) and the combined areas of inflammation within these sections were measured by video microscopy. Percent total area involved with inflammation (top panel) was calculated as [(total area of inflammation/ total lung area surveyed) 3 100]. The mean values for nondiabetic Ctrl mice (filled circles) and for STZc mice (open circles) diabetes are indicated by horizontal lines. The number lesions of any size visible at 320 magnification on H&E stained lung sections was counted (bottom panel). Horizontal lines indicate mean values for control and STZc groups. pp , 0.05. expression of CCL2, the lungs of STZc mice had lower levels of the might also be explained by an effect of DM to inhibit DC trafficking macrophage and DC chemoattractant CCL5. Jang et al. (25) re- to the LNs, we found no difference in the frequency of migrating

ported that M. tuberculosis induces expression of CCL5 mRNA and DCs of STZc or control mice stimulated with LPS. The response of by guest on September 26, 2021 protein by macrophages, whereas Salam et al. (26) showed that DC to M. tuberculosis and LPS surely differs in many ways but CCL5 contributes to host protective DC activation against M. tu- Khader et al. (18) reported that tracheal instillation of LPS or berculosis in vivo. We posit that in STZc mice, alveolar macrophages infected aerogenically with M. tuberculosis have a relative defect in generating signals, including CCL2 and CCL5, that recruit naive macrophages and DCs to the site of infection. DCs are thereby delayed in acquiring M. tuberculosis and consequently late in delivering Ag to the LNs despite intact capacity for LN homing. Although late delivery of M. tuberculosis from the lung to the LNs

FIGURE 6. Anti-PPD immunohistochemistry of lung sections from diabetic (STZc) and Ctrl mice with TB. Mice were infected with ∼100 CFU M. tuberculosis Erdman by aerosol and then lungs were isolated FIGURE 7. Pulmonary chemokines in diabetic and Ctrl mice with TB. 15 d later for immunohistochemistry with H&E counterstain. M. tuberculosis- Mice were infected with M. tuberculosis Edrman and lung lysates were infected macrophages are distinguished by brown staining (original magnifi- prepared 15 d p.i. for analysis by ELISArray of samples pooled from 4 mice cation 3400). per group (A), or by ELISA of samples from individual mice (B). pp , 0.05. The Journal of Immunology 6281 irradiated M. tuberculosis increased DC trafficking to the draining Disclosures LNs at a comparable level. These results, and our finding that ex- The authors have no financial conflicts of interest. pression of class II MHC, CD40, CD80, and CD86 on migrating DCs are not influenced by DM, points to a delay in DCs acquiring M. tuberculosis from infected macrophages as a key factor regu- References 1. Hull, M. W., P. Phillips, and J. S. Montaner. 2008. Changing global epidemi- lating the kinetics of adaptive immunity in DM. ology of pulmonary manifestations of HIV/AIDS. Chest 134: 1287–1298. Adverse effects of DM on TB defense in rodent models have been 2. Jeon, C. Y., and M. B. Murray. 2008. Diabetes mellitus increases the risk of active confirmed by other laboratories. Saiki et al. (27) and Yamashiro et al. tuberculosis: a systematic review of 13 observational studies. PLoS Med. 5: e152. 3. Shaw, J. E., R. A. Sicree, and P. Z. Zimmet. 2009. Global estimates of the (28) used high-dose i.v. infection of STZ-treated mice and found prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. 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Vallerskog et al., Delayed TB Immunity in Diabetic Mice

FIGURE S1. Levels of HbA1c, in whole blood. Blood from non-diabetic control mice

(filled circles) and STZc mice (open circles) was collected from the inferior vena cava 3 mo after STZ injection and prior to aerosol Mtb challenge. %HbA1c was determined by affinity chromatography. * p < 0.001.

FIGURE S2. Lung and thoracic LN leukocyte populations. Cells from non-diabetic control mice (Ctrl, filled bars) and STZc mice (empty bars) were collected from lungs

(A) and lymph nodes (B) at indicated time points and stained with mAb for analysis by flow cytometry. Briefly, cells were incubated 10 min with Fc-block Ab (anti-

CD16/CD32; BD Bioscience, San Jose, CA) followed by 30 min incubation with mAb

(all purchased from eBioscience, San Diego, CA) as follows: lung leukocytes were stained with F4/80, Gr-1, CD19, CD4, and CD8; LN cells were stained with CD19,

CD11c, CD4, and CD8. All cells were washed and fixed with BD Cytofix/cytoperm (BD

Bioscience) before analysis. Data analysis was performed with FlowJo v.7.2.5 software

(Tree Star Inc., Ashland, OR). The number of cells in each population of lung leukocytes was calculated from the frequency of cells according to flow cytometry multiplied with the number of cells in the lungs at each time point indicated. FIGURE S3. Anti-PPD immunohistochemistry of lung sections from diabetic (STZc) and control (Ctrl) mice with TB (n=3). Mice were infected with ~100 CFU Mtb Erdman by aerosol and then lungs were isolated (A) 21 d and (B) 28 d later for immunohistochemistry with H&E counter stain. Mtb-infected macrophages are distinguished by brown staining (X 400 magnification).

FIGURE S4. Pulmonary chemokines in diabetic (STZc) and control (Ctrl) mice with TB.

Mice were infected with Mtb Edrman and lung lysates were prepared (A) 12 d p.i. and (B)

28 d for analysis by ELISArray of samples pooled from 4 mice per group