Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment

Abstract

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-β, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.

Immunophenotyping of Peripheral Blood, Lymph Node, and Bone Marrow T Lymphocytes During Canine Leishmaniosis and the Impact of Antileishmanial Chemotherapy

“…Nonetheless, it should be noted that both protocols resulted in an increase of CD4 + Treg cell frequencies in all tissues, possibly in order to significantly reduce the frequency of CD8 + CD25 − FoxP3 − T cells present and to control the local inflammatory immune responses. Lastly, with respect to the effectiveness of either treatment, despite not being the scope of this work, the recovery of many subpopulations was achieved more quickly with the Megl+Al protocol than with the Milt+Al protocol, which is in agreement with previous results ( 66 ).…”

“…CD8 + Treg cell frequencies were also significantly increased in the blood and bone marrow, showing a possible action on immune responses mediated by CD4 + T cells, which can lead to parasite tolerance and disease progression. In the present work, the administration of either treatment protocol led to an overall recovery of the T cell subpopulations by the end of observation, reflecting the clinical improvement of the dogs ( 66 ). Nonetheless, it should be noted that both protocols resulted in an increase of CD4 + Treg cell frequencies in all tissues, possibly in order to significantly reduce the frequency of CD8 + CD25 − FoxP3 − T cells present and to control the local inflammatory immune responses.…”

Prognostic Factors and Life Expectancy in Canine Leishmaniosis

“…In terms of combined therapy, dogs treated with Me + A survived longer than those treated with M + A. However, these results must be interpreted with caution since, in general, veterinarians prescribe M + A [ 36 ] or A in monotherapy to dogs evidencing more clinicopathological alterations, and Me + A to animals that show fewer abnormalities [ 36 ], in order to minimize renal damage. The selection of pharmacological therapy with a low renal impact, particularly in dogs with previous renal dysfunction, is essential [ 26 , 75 ].…”

“…Therapy with anti- Leishmania drugs promotes the clinical cure, increases life expectancy [ 22 , 34 , 35 , 36 ] and decreases canine infectiousness to sand fly vector [ 35 , 37 , 38 , 39 , 40 , 41 , 42 ]. Meglumine antimoniate in combination with allopurinol (Me + A) is considered the “gold standard” treatment in the absence of severe clinical conditions [ 43 , 44 ], whereas miltefosine plus allopurinol (M + A) or allopurinol alone (A) are alternative treatments that are available for oral administration [ 45 , 46 ].…”

Comparison of Two Dosing Regimens of Miltefosine, Both in Combination With Allopurinol, on Clinical and Parasitological Findings of Dogs With Leishmaniosis: A Pilot Study

“…Despite its proven efficacy, MA has some drawbacks, including the high costs, the parenteral administration and the numerous reported side effects (18). MIL used at the registered dosage of 2 mg/Kg/die in association with allopurinol, is a good alternative (19)(20)(21)(22). Indeed, unlike MA, this molecule has a low impact on renal function and can be used in dogs with proteinuria, one of the most frequent clinicopathological alteration reported in CanL (23,24).…”