Pulmonary disease remains a leading cause of morbidity and mortality in HIV-infected individuals, with Pneumocystis pneumonia (PCP), one of the most common AIDS-defining opportunistic infections in the United States.1–4 In addition, the number of HIV-uninfected individuals at risk for PCP has grown because of increased use of immunosuppressive therapies.5,6
Because there are no Pneumocystis (Pc) vaccines available, current therapies and prophylaxis for PCP are restricted to chemotherapeutic agents. Trimethoprim–sulfamethoxazole (TMP-SMX) remains the most widely used antimicrobial agent for treatment of PCP and prophylaxis because of its safety, efficacy, and low cost.7 TMP-SMX is recommended as first-line prophylaxis against PCP in HIV-infected individuals with CD4+ T-cell counts of less than 200 cells per microliter, those with oral candidiasis and those with PCP after completion of PCP treatment regimen.8–10 Pc prophylaxis is also recommended for HIV-uninfected persons receiving immunosuppressive medications or who have an underlying acquired or inherited immunodeficiency.11,12
Recent studies have focused on the epidemiology and clinical consequences of Pc colonization, which is defined as the detection of Pc in respiratory samples that may occur in subjects with or without the symptoms of acute infection.13–15 Pc colonization is associated with low organism burden in respiratory samples and because Pc cannot be cultured in the laboratory, detection is accomplished using polymerase chain reaction (PCR)-based assays of respiratory samples.16–18 The prevalence of Pc colonization is variable among HIV-infected individuals, with reported rates ranging from 20% to 69%,2,3,19–22 even among those receiving anti-Pc prophylaxis and those with high CD4+ T-cell counts who are receiving antiretroviral therapy (ART).3,13 In the general population, Pc colonization rates may be higher than previously believed,23 and it is likely that Pc-colonized persons serve as a reservoir for transmission of Pc in PCP cases as well.24 Pc colonization has been reported in infants,25 persons receiving immunosuppressive therapies,26 health care workers,27 pregnant women,28 and persons with underlying pulmonary disease.26,29
Colonization with Pc may have important clinical implications, in addition to its contribution to transmission or development of PCP. In particular, several recent studies have focused on the role of Pc colonization and the development of chronic obstructive pulmonary disease (COPD).30–33 Pc colonization is associated with worse airway obstruction, increased risk of airway obstruction,31 and COPD in HIV-infected individuals,31,32,34 independent of smoking history or corticosteroid use.32 Other studies have reported increased systemic inflammation, including higher levels of interleukin (IL)-6, IL-8, and tumor necrosis factor-α associated with Pc colonization in COPD.35 Furthermore, in experimental animal models, Pc colonization is associated with obstructive lung disease and emphysema.36–38 In a study using an immunocompetent rat model, increased physiologic and anatomic emphysematous changes were reported in animals exposed to cigarette smoke in combination with Pc, compared with either alone.38 In a nonhuman primate (NHP) model of HIV infection, Pc colonization resulted in development of airway obstruction, radiographic emphysema, and enlargement of lung airspaces.36
To understand the relationship between Pc colonization and the development of HIV-associated COPD, our laboratory has developed a NHP model of naturally acquired Pc infection, in which macaques become persistently colonized with Pc after simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV)-infection.36,39,40 Susceptibility to Pc colonization in this model is associated with low plasma anti-Pc antibody titer at baseline and CD4+ T-cell levels of less than 500 cells per microliter after viral infection.39,40 Pc colonization in SHIV-infected macaques correlated with declining pulmonary function and increased pulmonary inflammation, compared with monkeys infected with SHIV alone.36,37,40–42 Because persistent Pc colonization has been noted in HIV-infected individuals despite Pc prophylaxis and colonization is associated with COPD, we sought to determine the effect of TMP-SMX treatment on established Pc colonization in SHIV-infected macaques. In addition, we tested whether reduction in Pc colonization improved pulmonary function in the macaque model of HIV-associated COPD.
MATERIALS AND METHODS
Before the initiation of this study, all animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Animal husbandry and experimental procedures were conducted in accordance with standards set forth by the Guide for the Care and Use of Laboratory Animals43 and the provisions of the Animal Welfare Act.44
Adult cynomolgus macaques (Macaca fascicularis, n = 16) were inoculated intravenously with 1 × 104.9 TCID50 of SHIV89.6P,45 which induces CD4+ T-cell lymphopenia and AIDS-like disease, including wasting and opportunistic infections.45,46 To promote Pc transmission, SHIV-infected macaques were cohoused with SIV- or SHIV-immunosuppressed, Pc-colonized macaques, which served as a source of Pc.39,40 Determination of Pc colonization status was performed by the detection of Pc DNA in bronchoalveolar lavage (BAL) fluid samples by nested PCR of the mitochondrial large subunit rRNA gene (mtLSU),41,47 and by anti-Pc serology using recombinant Pc kexin protein as the target antigen.39,40 Pc colonization was defined as positive nested PCR of BAL fluid and/or at least a 3-fold increase in plasma anti-Pc kexin (KEX1) titers.39,40 Study design is shown in Figure 1. For BAL fluid collection, a pediatric fiberoptic bronchoscope was directed into the right primary bronchus and wedged into a distal subsegmental bronchus that approximated the diameter of the bronchoscope. Four 10-mL aliquots of 0.9% saline were instilled and aspirated; fluid from a single animal was pooled. BAL fluid and peripheral blood were processed as described previously.36,39,41,42
At 25 weeks post-SHIV infection, 11 macaques were Pc colonized and 5 remained Pc negative. At this time, 7 of the Pc-colonized macaques (randomly selected from Pc-colonized group of 11 animals), and all of the Pc-negative animals were placed on TMP-SMX (TMP: 20 mg/kg and SMX: 100 mg/kg, daily, administered orally, confirmed by direct observation).48 Four Pc-colonized macaques remained untreated for the duration of the study (72 weeks post-SHIV infection).
Flow Cytometry Analysis of Peripheral Blood Cells
Peripheral blood samples were collected from macaques as described in the Supplemental Digital Content (see https://links.lww.com/QAI/A471). Peripheral blood leukocytes were counted, stained, and fixed for analysis by flow cytometry, as described.42 Antibodies used were mouse antimonkey CD3–FITC (clone SP34), mouse antimonkey CD4–allophycocyanin (clone L200), and mouse antimonkey CD8-PacificBlue (RPA-T8) (BD Pharmingen, San Diego, CA). Acquisition was performed using BD FacsDiva software on BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Forward-/side-scatter dot plots were used to gate the live lymphocyte population. All analyses were performed using FlowJo flow cytometry analysis software (Tree Star, Ashland, OR). Antech Diagnostics (Lake Success, NY) performed differential cell counts, and lymphocyte counts were used to determine absolute numbers of CD4+ T cells.
Pulmonary Function Testing
Pulmonary function tests were performed at baseline and monthly after SHIV infection using whole-body plethysmography49 and forced deflation technique36 to assess airflow obstruction. Briefly, animals were anesthetized with intravenous propofol [7.5–12.5 mg/kg (body weight)], and 2% lidocaine was given before intubation (3.5-mm endotracheal tube). Endotracheal tube placement was verified by a chest radiograph. Pulmonary function testing was performed using a Buxco whole-body plethysmograph (Buxco Electronics, Inc., Sharon, CT), and the BioSystems for Maneuvers Software (Buxco Electronics, Inc.) was used to collect data on flow rates and volumes. When 3 measurements for forced vital capacity were within 10% of each other, tests were considered valid.
Pc Kexin Antibody End Point Titer Determination
Reciprocal end point titers (RETs) to the Pc kexin-like protease (KEX1) were determined by enzyme-linked immunosorbent assay as previously described.39,40 Serial dilutions were made to determine end point titers, and goat antimonkey horseradish peroxidase was used for detection. The RET was calculated as the highest dilution at which the optical density values for the test sample were the same or less than an uninfected, Pc-negative control sample.
Statistical analyses were performed using Prism software or InStat software (GraphPad, La Jolla, CA). T tests and 1-way analysis of variance (ANOVA) were performed on ranked data.50 For each group of animals, comparisons between baseline values and values at other time points were made using a paired Student t test. Comparisons between groups of animals at a single time point were made using unpaired Student t tests. When comparing Pc-colonized and Pc-negative monkeys over multiple time points, 2-way repeated measures (RM) ANOVA was used for comparison. Fisher exact test was used to evaluate reduction of PCR+ samples after TMP-SMX treatment. A P value of <0.05 was considered significant.
Pc Colonization of SHIV89.6P-Infected Macaques
SHIV infection of macaques resulted in peak plasma viral load at 1–2 weeks after infection and rapid peripheral blood CD4+ T-cell decline (Fig. 2). Monkeys were exposed to natural transmission of Pc by cohousing with SIV+/Pc+ macaques, as described.39,40 By 8 weeks post-SHIV infection and Pc exposure, 4 of 16 monkeys had detectable Pc in BAL fluid by nested PCR. By 16 weeks post-SHIV infection, 11 of 16 macaques became Pc colonized and 5 macaques remained Pc-negative for the duration of the study (72 weeks).
Peripheral blood CD4+ T-cell levels (cells/μL) were monitored monthly after infection, and no significant difference in absolute number was observed in Pc-colonized (n = 11) and Pc-negative (n = 5) animals (Fig. 2A; P = 0.17, RM ANOVA). Additionally, peripheral blood peak viral loads were not significantly different between Pc-colonized and Pc-negative macaques (Fig. 2B; P = 0.35, unpaired t test).
Pulmonary Function in SHIV-Infected Macaques
Pulmonary function was measured at baseline and at monthly intervals after SHIV infection. Pc-colonized macaques exhibited significant declines in peak expiratory flow (PEF, P = 0.001) and FEV0.4 (forced expiratory volume in 0.4 seconds, P = 0.001) between baseline and 25 weeks post-SHIV infection (WPI) (Figs. 3A, C; paired t test), indicating pulmonary obstruction. No significant changes in pulmonary function (baseline vs. 25 WPI) were observed in Pc-negative macaques (Fig. 3; PEF; P = 0.21 and FEV0.4; P = 0.21, paired t test). These results confirm previous studies that showed that Pc colonization is associated with the development of COPD in SHIV-infected macaques and that pulmonary function deficits occur early after detection of Pc colonization.36,40
TMP-SMX Treatment of SHIV-Infected Macaques
We next tested whether treatment with TMP-SMX–reduced Pc colonization in SHIV-infected macaques and whether reduction in Pc colonization restored pulmonary function or slowed decline in macaques with pulmonary obstruction. At 25 weeks after SHIV infection, macaques with persistent Pc colonization (n = 11) were randomly assigned to TMP-SMX–treated (n = 7) or untreated groups (n = 4) (Fig. 1). TMP-SMX treatment was initiated, and bronchoscopy was performed monthly for the detection of Pc by nested PCR. The percentage of positive nested PCR samples for all BAL fluid samples in each group (TMP-SMX–treated and untreated) was compared before (n = 7 BAL fluid samples per animal) and during TMP-SMX treatment (n = 9 BAL fluid samples per animal). TMP-SMX treatment significantly reduced the percentage of Pc-positive BAL fluid samples (1.6% of 63 total BAL fluid samples; P = 0.0004; Fisher exact test) compared with untreated group (33.3% of 36 total BAL fluid samples) (see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/A471). BAL samples from 6 of 7 TMP-SMX–treated monkeys were Pc negative at all time points for the duration of the treatment (47 weeks).
As a secondary indicator of Pc colonization, we determined anti-Pc KEX1 antibody titers in SHIV infected, Pc-exposed macaques pre- and post-TMP-SMX treatment. Plasma anti-KEX1 reciprocal end point (RET) antibody titers increased in SHIV-infected animals that became colonized with Pc (Fig. 4A). Before TMP-SMX treatment, there was no significant difference in the serial mean plasma KEX1 IgG titers in the group of Pc-colonized animals that were subsequently TMP-SMX–treated (n = 7) and the Pc-colonized animals that remained untreated (n = 4) (P = 0.51; RM ANOVA). After 25 weeks post-SHIV infection, KEX1 IgG titers continued to increase in the untreated group. In contrast, KEX1 plasma IgG RET was significantly reduced in the Pc-colonized, TMP-SMX–treated macaques, compared with the Pc-colonized untreated animals (P = 0.021; RM ANOVA). These data indicate a treatment response to TMP-SMX, suggesting that when Pc burden is reduced, circulating antibody titers decline in response. TMP-SMX treatment did not significantly alter circulating IgG RET in the Pc-negative animals (Fig. 4B; P = 0.35; RM ANOVA). Additionally, IgG RET was not different between Pc-colonized, TMP-SMX–treated and Pc-negative animals (Fig. 4C; P = 0.34; RM ANOVA) or between Pc-colonized untreated animals and Pc-negative monkeys (P = 0.47; RM ANOVA) after TMP-SMX treatment initiation.
Pulmonary Function in TMP-SMX–Treated and Untreated Pc-Colonized Macaques
Pulmonary function was evaluated post-TMP-SMX treatment to determine whether the observed pulmonary function declines were the result of a transient response to Pc colonization and reversible with reduction in Pc burden, or whether pulmonary function decline was permanent. Pulmonary function was monitored at monthly intervals for the remainder of the study (25–72 weeks post-SHIV infection). Interestingly, while there was no significant improvement in pulmonary function more than 40 weeks after treatment, pulmonary function did not continue to decline in either the TMP-SMX–treated [PEF (P = 0.29; Fig. 5A) and FEV0.4 (P = 0.46; Fig. 5C)] or untreated group [PEF (Fig. 5B; P = 0.39) and FEV0.4 (Fig. 5D; P = 0.39)]. PEF and FEV0.4 did not decline significantly from baseline values by 72 weeks post-SHIV infection in Pc-negative macaques [data not shown; P = 0.70 (PEF); P = 0.70 (FEV0.4)].
To examine the reversibility of pulmonary obstruction in SHIV-infected, Pc-colonized macaques, animals were treated with the bronchodilator albuterol at 43 weeks post-SHIV infection (18 weeks of TMP-SMX treatment in the treated group), with no significant improvement in PEF or FEV0.4 after treatment (see Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/A471).
Increasing evidence suggests microbial colonization is associated with COPD exacerbations, perhaps through amplification of pulmonary inflammatory responses to noxious agents such as cigarette smoke.32,36,37,40,51–54 Several studies have shown that Pc colonization is associated with the development or progression of COPD in HIV-infected and non–HIV-infected individuals,31,32,52 although a causal relationship is difficult to ascertain in clinical studies. In a SHIV-NHP model of HIV infection, we demonstrated that progressive declines in pulmonary function parameters followed Pc-colonization, and monkeys infected with virus alone maintained normal lung function.36 Here, we show that decline in pulmonary function occurs early after Pc colonization and that Pc-induced obstructive changes are not reversible after reduction of Pc colonization with TMP-SMX or albuterol treatment, indicating development of COPD-like disease in these animals.
In the SHIV model, Pc colonization occurs by natural transmission as early as 2–4 weeks after viral infection coincident with CD4+ T-cell decline. Pulmonary function decline was observed as early as 4 weeks after initial evidence of Pc colonization, with all Pc-colonized animals exhibiting significant pulmonary obstruction within 1–4 months of Pc colonization. Pulmonary function did not change significantly from baseline levels in SHIV-infected, Pc-negative animals, supporting previous findings that pulmonary function deficits in this model were not a consequence of virus infection alone.36,40 We found no evidence of more profound SHIV infection in the Pc-colonized/COPD+ macaques based on the peripheral blood CD4+ T-cell levels or viral load, compared with Pc-negative monkeys with normal lung function, suggesting that decreased pulmonary function in the SHIV/Pc-colonized monkeys was not the direct result viral burden or more advanced AIDS. Previous studies showed that susceptibility of SHIV-immunosuppressed macaques to Pc colonization was associated with low baseline plasma anti-Pc antibody titers, suggesting a role for humoral immunity in control of Pc colonization and prevention of Pc-related COPD in this model.40 SHIV-infected macaques withheld from TMP-SMX treatment remained persistently Pc colonized although they did not develop PCP during the study period. This is likely due to the transmission of Pc from colonized macaques rather than macaques with PCP (K.A. Norris, unpublished data).
Pc colonization is common in HIV+ subjects.2,18,32 Reported incidence of Pc colonization varies, likely due to differences in patient populations examined, samples collected and detection methods used. There may be substantial differences in colonization prevalence, for example, between samples collected from oropharyngeal washes versus BAL fluid. Additionally, the relationship between Pc colonization and CD4+ T-cell counts is debated.3,55 It has been demonstrated that individuals with COPD are more likely to be Pc colonized compared with healthy smokers, and the frequency of Pc colonization is associated with worse pulmonary obstruction in HIV-infected3 and HIV-uninfected smokers.56 In HIV-uninfected persons, studies have demonstrated that Pc colonization is a risk factor for more severe COPD, independent of smoking history or corticosteroid use.32 The current study supports the concept that Pc colonization is associated with obstructive changes in a primate model of HIV infection and demonstrates that obstructive changes occur within weeks of initial Pc colonization in the macaque model of HIV infection.
Although there is substantial evidence to indicate that TMP-SMX is effective in preventing and treating PCP,8,57,58 there have been limited studies on the effects of TMP-SMX prophylaxis on Pc colonization in HIV+ individuals.3 The present study demonstrates that it is possible to reduce Pc colonization by aggressive treatment with TMP-SMX, as indicated by reduced detection by PCR and decline in anti-Pc antibody titers. Nevertheless, continuous treatment did not improve lung function, suggesting that structural damage of the lung parenchyma, previously shown to be associated with Pc colonization36 occurs as early as 6 months post-Pc exposure. Furthermore, no improvement in lung function was seen after bronchodilator treatment in the SHIV-infected/Pc-colonized macaques (see Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/A471). We previously demonstrated that in addition to worse pulmonary function, SHIV-infected, Pc-colonized macaques had increased anatomic emphysema compared with macaques infected with SHIV alone.36 Taken together, these results support the conclusion that Pc colonization induces irreversible changes in pulmonary function rather than transient, inflammation-mediated airway hyper-responsiveness.
Interestingly, while TMP-SMX treatment and reduction in Pc burden did not improve pulmonary function, we did not see further decline in untreated, Pc-colonized monkeys. At 72 weeks post-SHIV infection, parameters of pulmonary function, PEF and FEV0.4 were similar to values recorded before TMP-SMX treatment initiation (25 WPI) in the treated, Pc-colonized group. These data suggest that the initial damage associated with Pc infection, which occurs early after Pc colonization is not sustained at the same rate throughout infection, but is characterized by an initial sharp decline in pulmonary function, which is maintained, but does not decline further. As the kinetics of Pc colonization and development of COPD are difficult to assess in human populations, the present studies underscore the use of the NHP model for examining the consequences of Pc colonization at its earliest measurable time points.
It is interesting to note that Pc colonization results in early decline in pulmonary function in SHIV-infected macaques, but pulmonary function decline does not continue as typically occurs in human COPD. The development and progression of human COPD is multifactorial with genetic and extrinsic factors, such as cigarette smoke, contributing to pathogenesis.59,60 The combination of Pc colonization and smoking is associated with increased frequency of COPD and worse pulmonary function compared with that of non–Pc-colonized individuals.32 The role of Pc colonization, as well as other respiratory pathogens, in amplifying the host inflammatory response to cigarette smoke and other noxious agents has been proposed as a “vicious circle hypothesis.”54 In the context of NHP SHIV infection, Pc colonization is sufficient to induce COPD, but it may be that the absence of a “second hit” such as cigarette smoke precludes further decline in pulmonary function. Additionally, the number of studies in HIV-infected persons has found an association between respiratory symptoms, airway obstruction, and ART.34,61,62 The mechanism linking ART use with airway obstruction is not known, however, immune reconstitution inflammatory syndrome associated with ART initiation may result in a chronic inflammatory response, which may exacerbate COPD pathogenesis.62 The NHP model of HIV-associated COPD is a valuable resource that should allow for direct assessment of the influence of extrinsic factors such as smoking, ART, or illicit drugs on disease progression.
The relationship between Pc colonization and COPD development may be the result of chronic inflammatory changes that occur in the alveoli in response to Pc persistence. We have previously shown increased levels of proinflammatory mediators (IL-1b, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and Th2-type cytokines (IL-4, IL-5, and IL-13) in BAL fluid of SHIV-infected macaques after Pc colonization compared with macaques infected with SHIV alone.36 Peak levels of these mediators occurred by 20 weeks post-SHIV/Pc exposure, supporting a role for inflammation in the early response to Pc colonization and development of COPD. Many of the inflammatory changes reported in Pc infection, including influx of CD8+ T cells and neutrophils and increased IL-8 production are similar to inflammatory profiles associated with COPD.63–66
The data presented here suggest TMP-SMX treatment may mitigate Pc colonization in a NHP model of HIV infection; however, damage to the host lung, likely resulting from host immune responses to Pc colonization, occurs early after colonization and cannot be reversed with chemotherapeutic treatment. While TMP-SMX has been shown to effectively prevent PCP in immunocompromised hosts, prolonged TMP-SMX therapy would likely have little effect in preventing or improving Pc-induced COPD. Several studies have explored the development of prophylactic Pc immunization to protect against PCP.67–70 The relationship between Pc colonization and the development of permanent obstructive lung damage in at-risk populations, such as HIV+ individuals, and the lack of efficacy of TMP-SMX treatment in preventing Pc colonization or COPD, as demonstrated in this model, supports the rationale for expanding such vaccine development to include prevention of Pc colonization and obstructive lung disease.
The authors thank Dr Chris Janssen for excellent veterinary care and Dr Kurtis Moseley for consultation regarding statistical analyses.
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