A Novel Partial MHC Class II Construct, DRmQ, Inhibits Central and Peripheral Inflammatory Responses to Promote Neuroprotection in Experimental Stroke

Abstract

Recognizing that the pathologic progression of stroke is closely associated with aberrant immune responses, in particular the activation of peripheral leukocytes, namely T cells, we hypothesized that finding a treatment designed to inhibit neuroantigen-specific Tcells and block cytotoxic monocytes and macrophages may render therapeutic effects in stroke. We previously reported that subcutaneous administration of partial MHC class II constructs promote behavioral and histological effects in stroke mice by centrally promoting a protective M2 macrophage/microglia phenotype in the CNS and peripherally reversing stroke-associated splenic atrophy. Here, we employed a second species using adult Sprague-Dawley rats exposed to the middle cerebral artery occlusion stroke model and observed similar therapeutic effects with a mouse partial MHC class II construct called DRmQ, as evidenced by reductions in stroke-induced motor deficits, infarcts, and peri-infarct cell loss and neuroinflammation. More importantly, we offered further evidence of peripheral sequestration of inflammation at the level of the spleen, which was characterized by attenuation of stroke-induced spleen weight reduction and TNF-ɑ and IL-6 upregulation. Collectively, these results satisfy the Stroke Therapy Academic Industry Roundtable criteria of testing a novel therapeutic in a second species and support the use of partial MHC class II constructs as a stroke therapeutic designed to sequester both central and peripheral inflammation responses in an effort to retard, or even halt, the neuroinflammation that exacerbates the secondary cell death in stroke.

Introduction

Cerebral ischemia remains as a leading cause of death worldwide [1, 2]. Neuroinflammation is a major secondary cell death process [3, 4]. The short therapeutic window limits the number of available stroke treatments [5–7]. Immune-targeted therapeutics may attenuate stroke neuroinflammation [8, 9]. Inflammatory response occurs both centrally and peripherally, with the spleen playing a critical role in secondary cell death [10]. Early immune expansion by way of partial MHC II construct may abrogate ischemic-induced inflammation [11, 12]. Regulatory switching of T cell cytokine expression may modulate both splenic and cerebral inflammatory pathways [13]. Recombinant T cell receptor ligand, RTL1000, reduces infarct volume in the absence of tPA, while offering additional neuroprotection in combination with tPA [14]. MHC II constructs stand as potent stroke therapeutics due to their neuroantigen-specific modulation of T cells [11, 12, 15].

MHC class II construct, DRa1-mMOG-35–55 (DRa1 domain covalently linked to mouse (m)MOG-35–55 peptide), can alleviate cell death in stroke and traumatic brain injury [12]. We recently produced more potent second-generation constructs, DRhQ (DRa1_L50Q-human (h)MOG-35–55) for clinical development [15] and DRmQ (DRa1_L50Q-mMOG-35–55) that is highly effective for treating experimental autoimmune encephalomyelitis in mice [16, 17]. Here, for the first time, we tested DRmQ in a rat model of stroke. In order to effectively translate this drug into the clinic, Stroke Therapy Academic Industry Roundtable (STAIR) criteria must be followed to further advance this stroke therapy to stroke patients [18]. In particular, the use of at least two different species of stroke model is necessary to fully assess clinical relevance of MHC II constructs. In this study, we tested the anti-inflammatory effects of DRmQ in the rat model using established stroke behavioral and histological assays. We hypothesized that DRmQ targeted both central and peripheral inflammatory responses, including the attenuation of splenic inflammation, as an effective means of conferring stroke neuroprotection.

Methods

Subjects

All experimental procedures were approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley rats (8 weeks old, N = 20) were housed under normal conditions, two per cage (20 °C temperature, 50% relative humidity, and 12-h light/dark cycle) (Fig. 1a).

Fig. 1.

Procedural timeline. Specific timepoints are provided for the experimental design and procedures. Behavior tests: motor activity measured by rotarod (a), paw grasp (b), forelimb akinesia (c), and balance beam (d). The DRmQ treatment group rats showed significant improvement on day 3 (*p < 0.05, **p < 0.01). Nissl staining for vehicle and DRmQ treatment groups. a, c, f–h Quantitative analysis of the infarct volume in vehicle and the DRmQ treatment group. Coronal brain sections showed infarct area (black dashed line). The DRmQ treatment group showed significantly reduced infarct volume (*p < 0.05). The chart showed the percentage of contralateral volumes in the cortex, striatum, and hemisphere. The DRmQ treatment group decreased the volume (****p < 0.001). b, d, and e Quantitative analysis of live cells in the peri-infarct area (cortex and striatum). Arrowheads indicate the live cells. The graph represents the ratio between live cells in ipsilateral and contralateral regions of the brain lesion. The live cells in the peri-infarct area cortex (**p < 0.01) and striatum (*p < 0.05) were increased in the DRmQ treatment group. Scale bar = 50 μm

Stroke Surgery

Stroke surgery was performed using the middle cerebral artery occlusion (MCAo) technique as described elsewhere [19]. Rats that reached 80% cerebral blood flow reduction during occlusion were used in this study. All animals survived up to day 3 post-MCAo.

Drug Administration

At 4-h post-MCAo, randomly selected animals were injected subcutaneously with either 0.2 cc of 1 mg DRmQ or vehicle (n = 10 per treatment group).

Behavioral Tests

All investigators testing the animals were blinded to the treatment condition. Each rat was subjected to a series of behavioral tests before MCAo (baseline), at day 1 post-MCAo, and before perfusion (day 3). The tests included rotarod, elevated body swing test (EBST), balance beam, forelimb akinesia, and paw grasp tests, with detailed protocols described elsewhere [19, 20].

Euthanasia and Perfusion

Under deep anesthesia, rats were euthanized on day 3. For immunohistochemistry, brain and spleen tissues were processed for analyses of inflammation using rabbit polyclonal anti-tumor necrosis factor alpha (TNF-α; 1:500; ab6671) and rabbit polyclonal anti-IBA1 antibody (IBA1; 1:500; Wako; 019–19741) counterstained with DAPI (Vector Laboratories, USA). Coronal sections were examined using a confocal microscope (Zeiss). Control studies included exclusion of primary antibody substituted with 5% normal goat serum in 0.1 M PBS. No immunoreactivity was observed in these controls. Alternate tissue sections were processed for Nissl staining (0.1% cresyl violet solution; Sigma-Aldrich) using a standard protocol to evaluate the peri-infarct injury (NIH Image Software, USA).

Spleen Assay

Spleen weight was measured as previously described [16, 21, 22]. Additionally, the spleen was processed for ELISA. Concentrations of inflammatory cytokines (IL-6, TNF-α) in spleen were detected using ELISA kits (Abcam, USA).

Statistical Analyses

The data were evaluated statistically using paired t test with statistical significance preset at p < 0.05. (GraphPad version 5.01). Normality test via Kolmogorov-Smirnov test revealed that values were less than 5% of the critical values.

Results

DRmQ Ameliorates Stroke-Induced Behavioral Deficits

DRmQ-treated group displayed significantly improved functional outcomes in all motor tests examined here. Rotarod test showed the DRmQ-treated stroke animals had significantly prolonged time spent by the animal to maintain balance on the rod (**p < 0.01) compared with vehicle-treated stroke animals (Fig. 1b). Similarly, paw grasp, forelimb akinesia, and balance beam revealed that the DRmQ-treated stroke animals displayed significantly lower stroke severity scores (*p < 0.05, **p < 0.01) compared with vehicle-treated stroke animals (Fig. 1c–e).

DRmQ Reduces Cerebral Infarcts and Peri-Infarct Cell Loss

Compared with the vehicle-treated stroke animals, the DRmQ-treated stroke animals presented with a smaller cerebral infarct area (**p < 0.01) (Fig. 1f, magnified in g), as well as a significant reduction in cell loss (albeit, improved cell survival) in the peri-infarct cortex (**p < 0.01) and striatum (*p < 0.05), and significantly smaller infarct volume in cortex, striatum, and entire hemisphere compared with vehicle-treated stroke animals (****p < 0.001) (Fig. 1h–m).

DRmQ Dampens Stroke-Induced Inflammatory Response in the Brain

DRmQ-treated stroke animals exhibited significantly lower number of the pro-inflammatory cytokine TNF-ɑ positive cells in the peri-infarct area compared with the vehicle-treated stroke animals (****p < 0.001) (Fig. 2a). Similarly, DRmQ-treated stroke animals displayed a significant reduction in IBA1 positive activated microglial cells in the peri-infarct cortex (****p < 0.001) (Fig. 2b).

Fig. 2.

Immunohistochemical analysis of TNF-α and IBA1 in the peri-infarct area. Arrowheads indicate the positive cells. a Relative fluorescence intensity showed TNF-α (red) and DAPI (nuclear staining) (blue). The graph shows that the DRmQ treatment group had decreased cell count compared with the vehicle group (****p < 0.001). b Representative merged images counterstained with DAPI (nuclear staining) (blue). IBA-1 antibody was represented by the red color. Quantitative analysis demonstrated that the DRmQ treatment group had significantly reduced cells compared with the vehicle group (****p < 0.001). Scale bar = 100 μm. Spleen weight and ELISA analysis of TNF-α and IL-6 in the spleen. a Analysis of spleen weight showed heavier weights in the DRmQ treatment group compared with the vehicle group (**p < 0.01). b, c ELISA results showed decreased expression of TNF-α and IL-6 in the spleens of the DRmQ treatment group (**p < 0.01)

DRmQ Mitigates the Splenic Inflammatory Response

To determine the effects of DRmQ on the peripheral inflammatory response, the spleen was analyzed for weight and immunohistochemistry was performed. DRmQ-treated stroke animals reported higher spleen weights than stroke animals that received the vehicle (**p < 0.0) (Fig. 2c). Staining of stimulated splenocytes revealed significantly reduced levels of inflammatory cytokines TNF-ɑ (**p < 0.01) (Fig. 2d) and IL-6 (**p < 0.01) (Fig. 2e) in stroke animals that received DRmQ compared with vehicle-treated stroke animals, suggesting a suppression of the splenic inflammatory response.

Discussion

We demonstrated that DRmQ treatment ameliorated stroke-induced behavioral and histological deficits. In the brain, DRmQ significantly reduced the expression of the pro-inflammatory cytokine TNF-α and IBA1-activated microglia. This reduction in pro-inflammatory cytokines and microglia/macrophage activation may contribute to the DRmQ ability to reduce the secondary cell death in stroke. In a similar robust fashion, DRmQ attenuated inflammation in the spleen, a peripheral organ rich in inflammatory cells, as evidenced by decreased levels of IL-6 and TNF-α expression. Altogether, these findings showed that DRmQ is able to modulate the inflammatory response by sequestering pro-inflammatory cytokines centrally and peripherally in affording its neuroprotective properties.

Neuroinflammation has been the hallmark of secondary cell death in neurovascular diseases such as stroke and traumatic brain injury [10, 11, 14, 15, 23–25], thus targeting this post-injury inflammation is a promising therapeutic approach for neurological diseases. DRα1-mMOG-35–55 protects against stroke and other neurovascular diseases via modulation of inflammation [10, 12, 17]. In particular, DRa1-mMOG-35–55 significantly reduces the infarct size in both male and female wild-type C57BL/6 mice after MCAo by suppressing activated microglia and infiltrating monocytes [8], although females required a 10-fold higher dose of drug [16, 26]. DRa1-mMOG-35–55 has been also shown to exert similar therapeutic effects in the distal MCAo stroke model by reducing infarct size, modulating microglia polarization toward anti-inflammatory phenotype, and decreasing pro-inflammatory cytokines such as IL-1α and IL-17 [8, 12, 13].

The novelty of the present study is illustrated by two key observations: First, we advanced the translational platform of DRmQ by demonstrating its therapeutic effects in a second species along the lines of STAIR criteria [18]. Second, we offered further support to the important contribution of the peripheral component to the stroke pathology and its treatment, in that DRmQ also targeted the stroke-induced peripheral inflammation originating from the spleen, previously implicated as a critical therapeutic target for stroke neuroprotection [21, 22]. This robust anti-inflammatory property of DRmQ features its capacity to modulate the inflammatory response both centrally and peripherally, which likely contributed to the observed therapeutic behavioral outcomes.

Limitations of the present study include the need for a long-term study to examine whether the therapeutic effects could extend into the chronic phase of stroke. Although we did not detect any overt harmful behavioral and histological outcomes arising from the DRmQ treatment, monitoring any potential adverse effects in the long-term may be of interest especially if chronic treatment of DRmQ is indicated to fully sequester the progressive inflammation in stroke. Optimization of routes of administration, as well as the range of effective and tolerable doses, is also warranted.

In conclusion, we replicated our previous study performed in mice [8, 15, 17, 26] using rats exposed to MCAo and demonstrated similar levels of neuroprotective effects with the mouse partial MHC II construct called DRmQ. We further showed that DRmQ possesses central and peripheral beneficial outcomes, effectively improving motor and neurological performance and reducing brain damage associated with stroke. In tandem with previous findings [21, 22], DRmQ robustly sequestered splenic inflammatory response. Because this MHC-independent effect circumvents the need for tissue typing, these preclinical findings should expedite the application of DRmQ (albeit its human homolog DRhQ) in humans with stroke. The present results thus satisfied a key translational enabling guideline of testing DRmQ in a second animal species, providing solid guidance in supporting the use of DRmQ for treatment of stroke and potentially other neurological disorders with underlying inflammation pathology.