Intrarectal

Intrarectal Administration of Clodronate Liposomes

Currently, a single paper cited intrarectal administration of clodronate liposomes [1]. Few details of the the instillation method were provided and it is unclear as to whether a polycarbonate tip on an automatic pipettor or a glass-capillary type micropipette was used for the instillation of the liposome suspension. The key questions relate to how long the suspension was retained in the colon and how far into the colon the suspension penetrated although the authors observe that macrophage depletion was limited to the descending colon. The authors do not report fasting the animals or eliminating fiber from the animal diet prior to dosing the clodronate liposomes as has been incorporated into other protocols for colitis [2]. Likewise, the effectiveness of the instilled liposome suspension would seem to be affected by the colon content at the time of dosing.

The appropriate volumes for enemas is reported to be 0.2 ml for mice and 1 ml for rats [2] although several papers use 0.1 ml doses in mice and as much as 3 ml in rats. We have not identified standard protocols for intrarectal injections in rodents, but many report holding the rodents in a vertical, head-down position for a minute or so post-intrarectal instillation. This would appear to be a reasonable addition to the method that should increase the efficacy of intrarectal delivery.

Many also utilize rubber or plastic tubing for delivering the liquids 2 cm or more beyond the rectum into the descending colon, or more specifically, an intracolonic instillation. Since the rectal tissue plays little or no role in the disease process, ensuring that the clodronate liposome suspension has maximal access to the colon may enhance and/or extend the depletion period. A study utilizing fluorescently- and/or visibly- (DiI incorporation accomplishes both) labelled liposomes could assist in the evaluation of the retention of the intrarectally-delivered liposomes as well as their distribution within the colon.

Colonic macrophages have been shown to demonstrate decreased phagocytic activity due to the ingestion of dextran sodium sulfate (DSS) [3, 4, 5]. The majority of the data in this paper was collected in animals which were dosed with clodronate liposomes on days -1, +1, +3 and +5; the animals were given water containing DSS beginning on day 0. This could mean that treatment with clodronate liposomes post-oral administration of DSS to mice may result in ineffective depletion due to limited uptake of clodronate liposomes by colonic macrophages which are non-phagocytic due to blockade by DSS, thus the surprisingly rapid repopulation (50% replenished by day 7) of macrophages even after 4 intrarectal treatments with clodronate liposomes. If DSS is dosed to the animals prior to any clodronate liposome treatment, would any MΦ depletion be observed? We believe that this is an important factor to consider.

Another contrary interpretation of the data in the Qualls, et. al. paper is that macrophage uptake of DSS may limit DSS-induced inflammation, therefore macrophage depletion may result in the colon tissues being exposed to higher levels of DSS.

Qualls, et. al. chose not to include empty liposome control groups in their studies due to the fact that the control liposomes induced “a partial reduction in the percentage of MΦ in the colon and could affect the physiology of the remaining colonic MΦ (data not shown…” We would argue that this is precisely the rationale for including empty liposome controls and that any results should be compared to those obtained with empty liposome controls. These control results may very well have elucidated some of the potential effects of macrophages in the disease. For example, does a reduction in the phagocytic capacity of macrophages (due to liposome uptake without associated toxicity ) and in the absence of macrophage destruction have any effect on the disease? MΦ blockade by control liposomes could reduce the uptake of DSS by the resident MΦ; this may reduce the initial inflammatory response since MΦ would not be able to phagocytose DSS or worsen the disease because uptake of DSS by resident MΦ lessens the DSS effect on colonic tissue. Any reduction in the number of MΦ in the colon or changes in MΦ morphology induced by control (empty) liposomes should have been a transient reaction since liposomes are not toxic to MΦ, but they can inhibit MΦ phagocytosis for several hours.

It is common to observe an increase in neutrophil infiltration into the pulmonary space post-clodronate, but not control, liposome treatment. Perhaps neutrophil infiltration as a result of clodronate liposome treatment is a factor in this model as well, but no clodronate-liposome treated animals in the absence of DSS-induced colitis were evaluated for neutrophil infiltration.

And, finally, a primary function of the colon is in providing a highly effective barrier around the colon. Therefore, it is possible that this barrier is impermeable to free clodronate and that clodronate released from liposomes or dead/dying macrophages could temporarily accumulate locally attaining concentrations which could effect surrounding cells (see Intrapulmonary Administration for detailed discussion). While clodronate sequestration seems most unlikely inside the colon, it remains a pertinent control since intracolonic clodronate administration has not been previously evaluated to our knowledge.

This paper illustrates the necessity of considering the effects of control and clodronate liposomes on the chosen model. Given that the model employed a particulate agent (DSS) which is phagocytosed by MΦ, detailed analysis of the effects of MΦ blockade by both DSS, clodronate liposomes and control liposomes must be performed. Since each of these particles will likely evoke MΦ blockade, any observed results may simply be due to which particle reached the MΦ first.

Wanatabe, et. al. utilized poly-lactic acid µspheres containing clodronate, dosed intrarectally, to deplete macrophages in an IL-10 deficient mouse model of colitis, therefore the only particle type affecting the MΦ was the clodronate µspheres [7]. Unfortunately, they did not assess the effects of control µspheres (without clodronate), but they did observe increased disease activity when the colonic MΦ were depleted. Their study was much less rigorous than the Qualls, et. al. study as they did not pursue the effects of neutrophil infiltration nor evaluate cytokine and chemokine production. However, their study results do suggest that the use of a particulate material in inducing the collitis, as Qualls, et. al.did, does not change the conclusion that colonic macrophages play a pivotal role in ameliorating acute colitis in these animal models.

More recently, Qualls et. al. reported that depletion of dendritic cells (DC) alone in CD11c-DTR mice suppressed DSS-induced colitis, but without the concomitant increase in CXCL1, neutrophil influx and MPO activity [8]. We believe that this result further presses the question of the role of clodronate liposomes (or clodronate liposome MΦ depletion) in neutrophil influx and activation. An initial, single dose of intrarectal clodronate liposomes for depleting resident MΦ/DC along with multiple intravenous doses of clodronate liposomes could perhaps accomplish colonic MΦ/DC depletion and prevent subsequent repopulation (by depleting circulating monocytes) allowing for colonic recovery from any potential effects of clodronate liposome MΦ depletion, such as neutrophil influx, should it occur. Induction of DSS-mediated colitis post-recovery would allow distinction of possible neutrophil influx and activation as a result of clodronate liposome depletion from the neutrophil effects due to the absence of colonic MΦ.

Again, we emphasize that although the goal of clodronate liposome-mediated macrophage depletion is usually to generate animals which are normal in every other way except that they are devoid of macrophages, there can be effects due to

Liposome administration…control groups treated with empty liposomes are necessary.
Clodronate administration…control groups treated with unencapsulated (free) clodronate are necessary.
Clodronate liposome administration…macrophages are not killed for several hours post-treatment and may not behave as untreated macrophages in the interim.
Macrophage death…even relatively “silent” apoptotic cell death will solicit other macrophages, immature monocytes or other cells (i.e. neutrophils) to clear cellular debris and these cells may “report” extensive cell death in order to recruit replacement mononuclear cells.

Therefore, experiments elucidating the effects of the process of macrophage depletion may need to be considered and incorporated into the study.

The authors further reported an increase in IL-6 as a result of DC depletion and suggested that IL-6 may be the key to the increased severity in colitis after DC depletion. While these authors stated that IL-6 is reported to inhibit neutrophil infiltration, at least one of their cited references exclusively discusses the proinflammatory effects of IL-6 on neutrophils and other cells [9]. (Two of the other references appear to focus on the anti-inflammatory effects of IL-6 in exercise-induced inflammation.) IL-6 or its mRNA levels were not evaluated in the earlier study in which clodronate liposomes were used to deplete MΦ/DC, but the authors reported in this 2009 paper that they observed an increase in IL-6 production when both MΦ and DC were depleted (unpublished data) although the mode of depletion was not specified. While we are not well read in the substantial literature on experimental colitis, we believe that clodronate liposome studies including the controls that we proposed earlier may be helpful in dissecting the contributions of the various inflammatory cells, cytokines and chemokines in this model.